The endocrine pancreas of spontaneously diabetic dbdb mice: microangiopathy as revealed by transmission electron microscopy

ELSEVIER

Diabetes Research and Clinical Practice 30 (1995) 89%100

The endocrine pancreas of spontaneously diabetic dbjdb mice: microangiopathy as revealed by transmission electron microscopy Mitsuo Nakamura”,*, Hirokazu Kitamura”, Sadanori Konishib, Masahiko Nishimura”, Junko Onod, Keisuke Ina”, Tatsuo Shimada”, Ryosaburo Takakif “Department

of Anatomy, School of Medicine, Oita Medical Universitv, Idaigaoka I-l Hazama-cho, Oita Prefecture, Japan bGraduate School of Mathematics, Kyushu University, Fukuoka City, Fukuoka Prejicture, Japan ‘Institute of’ Esperimental Animals, Hamamatsu University of Medicine, Hamamatsu City, Shizuoka Prefecture, Japan dClinical Chemistry and Laboratory Medicine, Fukuoka University School of Medicine, Fukuoka City, Fukuoka Preficture, Japan eSchool qf Nursing, Oita Medical University, Idaigaoka I-l Hazama-cho, Oita Prefecture, Japan rOita Medical University, Idaigaoka l-l Ha:ama-cho Oita Prefecture, Japan

Received 10 July 1995; revised 23 August 1995; accepted 27 September 1995

Abstract

Abnormalities in ultrastructures of islet capillaries were detected in dbjdb mice, with the visual inspection and morphometry of electron micrographs. The observed changes are: (1) capillary scarcity; (2) increase in the mean and diversity of capillary size; (3) pericapillary edema and fibrosis; (4) hypertrophy of the pericyte and abundance therein of actin-like microfilaments; and (5) luminal irregularity. Changes (2), (3) and (4) are conceived to indicate hyperperfusion, capillary hypertension and secondary vascular response. In particular, such pericyte changes were found to be shared by other organs whose capillaries are susceptible to diabetic complications. Diabetic microangiopathy; Spontaneous diabetes mellitus; Endocrine pancreas; Electron microscopy; Morphometry

Keywords:

1. Introduction Systemic abnormalities in the morphology of microvessels have been reported in spontaneous and experimental diabetes [ 1- 111. The pancreatic islet is among organs whose capillaries are

* Corresponding author.

affected in diabetes [2,11]. In our previous study [I 11, we have examined the pancreatic islet of alloxan-diabetic rats, and have detected the following ultrastructural changes: capillary basement membrane thickening, pericapillary edema and fibrosis, capillary scarcity, capillary narrowing, and intrainsular occurrence of arterioles. In studies with alloxan-diabetic animals, there exists the possibility that the toxic action of alloxan directly

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affects the morphology of capillaries. The primary purpose of this study is to examine electron microscopically the islet capillaries of dbjdb mice, an animal model of genetically inherited type 2 diabetes [12,13]. Among the present observations, there were unexpected hypertrophic and hyperfunctional changes of the pericyte. Whether similar changes are the casein other organs susceptible to diabetic complications was also examined. 2. Materials and methods 2.1. Animals

For examination of pancreatic islets, 11 dbjdb male mice and, as controls, 12 C57BL male mice were used. They were aged 4 months (three dbjdb and three C57BL males), 6 months (four dbjdb and five C57BL males) and 12 months (four db/ db and four C57BL males). For preliminary examination of the retina, cecum and sciatic nerve, a separate group consisting of six dbjdb mice aged 4 months (three males) and 6 months (one male, two females), and seven C57BL mice aged 4 months (two males, one female) and 6 months (one male, three females) were used. All these mice were grown in the Institute of Experimental Animals of Hamamatsu University of Medicine, under the following conditions: at 23°C 50% humidity, 12-h light/l2-h darkness. One to 2 weeks before experiment, they were transferred to the Animal Laboratory Center of Oita Medical University, where they were reared under similar condition until use. Prior to morphological examination, body weight was measured and blood samples were drawn from the tail vein for plasma glucose determination. Morphological examination was carried out using fed animals deeply anesthetized with Nembutal. 2.2. Electron microscopy

Piecesof the pancreas were resectedand immersion-fixed in our modified Dalton’s fixative [14], at 4°C for 2 h. For other organs to be examined, animals were perfusion-fixed with chilled Karnovsky’s fixative. Pieces of cecum, retina, sci-

atic nerve and kidney were resected and immersion-fixed in Karnovsky’s fixative at 4°C for 2 h, followed by postosmication. After fixation, tissue pieces were dehydrated and embedded in the epon-epoxy resin. Ultrathin sections were made, mounted on square-meshed No. 150 copper grids and stained in metabolic uranyl acetate and aqueous lead citrate. The sections were electron microscopically examined at 80 kV, at nominal magnifications ranging from 880 to 5000. Magnifications were calibrated biweekly. Electron micrographic negatives were enlarge-printed as desired. For the pancreatic islet, portions of the section limited by grid bars in order to meet the following criteria were selected to be examined: (1) the portion should contain areas of islet tissues as wide as possible; (2) at least a fragment of the portion should be occupied by exocrine pancreatic tissues. Electron micrographs were controlled by visual inspection (all the organs examined) and morphometry (pancreatic islet only). 2.3. Morphometry

The following variables were determined on electron micrographs of the pancreatic islets, either by visual counting or with the aid of an electronic curve-planimeter (Ushikata type 360i): the dimension of islet tissue examined (variable 1); the number of capillaries encountered within variable 1 (variable 2); internal endothelial circumference (variable 3); the area encircled by internal endothelial circumference (variable 4); external endothelial circumference (variable 5); and the total area of the pericyte(s) and its process(es) surrounding a capillary (pericyte area per capillary, variable 6). These variables, except for variable 2, were expressed in ,um or pm’. Among these variables, variables 5 and 6 were adopted as parameters to be appreciated. The following additional parameters for appreciation were calculated from these variables: islet capillary density, which was obtained by dividing variable 2 by variable 1; luminal irregularity index, which equals to variable 3/2,/ rr x variable 4; and the pericyte area per unit external endothelial circumference, which is variable 6/variable 5.

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Fig. 1. Islet tissues of 6- (A) and 12-months (B)-old C57BL mice containing a capillary (CAP). Attenuated (AS) and non-attenuated WA S) segmentsof endothelial cells (EC) are distinguished. Pericyte (P) and pericyte processes(PP) are apposed to the endothelium. The adluminal surface of the endothelium is rather smooth. RBC, erythrocyte; CL, capillary lumen. Fixed in modified Dalton’s fixative. x 6600.

2.4. Statistical methods

Animals were grouped according to strain and age. For each animal, four to six islet profiles and the capillaries in them were measured. Data were pooled for each group. The pooled data were statistically compared between different strains and different ages, using an unpaired t-test. If the P value was below 0.05 the difference was regarded as significant. For parameters other than islet capillary density, data were natural-logarithmically transformed before analysis, considering rightward skewness of distribution. At the transformation, zero values were omitted from the data.

3. Results 3.1. Body weight and blood glucose level

All dbjdb mice studied weighed markedly heav-

ier as compared with age-matched controls, without any overlaps (20-25 g for 4-month C57BL mice; 37-43 g for 4-month dbjdb mice; 28-30 g for 6-month C57BL mice; 45-59 g for 6-month dbjdb mice; 26-28 g for 12-month C57BL mice; 41-48 g for 12-month dbjdb mice). All the dbjdb mice exhibited intense hyperglycemia ( > 400 mg/dl), whereas C57BL mice were invariably normoglycemic ( < 200 mg/dl). 3.2. Electron microscopic jindings by visual inspection

The islet capillaries of C57BL mice were of fenestrated type, in accordance with the text book description [15]. Thus, a substantial portion of the endothelial cell cytoplasm assumed an attenuated shape and was provided with numerous fenestrae closed by diaphragm(s). The remaining cytoplasm of the cell was of appreciable thickness and contained nucleus and cell organelles (Fig. 1A and

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Fig. 2. Islet tissue of a dbjdb mouse aged 4 months containing two capillaries (CAP 1 and CAP 2). In one capillary (CAP l), the lumen is narrow and of intricated contour. Pericapillary space (PS) is wide and contains flocculous materials. EC, endothelial cell; PP, pericyte process. Fixed in modified Dalton’s Fixative. x 6600. Fig. 3. Islet tissue of a dbjdb mouse aged 6 months containing a dilated capillary (CAP). Wide pericapillary space (PS) contains flocculous materials and pericyte processes(arrow). Adluminal surface of the endothelium is uneven. Erythrocyte (RBC). Fixed in modified Dalton’s fixative. x 6600.

Fig. 1B). A few cytoplasmic elevations were found on the adluminal surface of this cell type (Fig. 1A and Fig. 1B). Pericyte(s) and/or its process(es) were apposed to its abluminal surface. The cell body of the pericyte was infrequently observed in C57BL mice. Pericyte(s) and its process(es) were found to contain small to moderate amounts of actin-like microfilaments and appreciable numbers of elements of cell organelles (Fig. 6). The microfilaments occasionally exhibited a tendency to cluster at the periphery of the cytoplasm (Fig. 6). A thin basement membrane was found to run beneath the endothelium and around the pericytes and their processes(Fig. 1A and Fig. 1B). We had the impression that pericytes were more frequently observed among older (6 and 12 months old) than younger (4 months old) C57BL mice. Otherwise, age-related differences were not evident in normal mouse islet capillaries.

In dbjdb mice, the following abnormalities of islet capillaries were observed; first, islet capillaries tended to be larger and more diverse in size than in C57BL mice. As a result, dilated and narrowed capillaries were occasionally encountered (Fig. 2, Fig. 3, Fig. 4). Second, capillary luminal surface appeared rather undulated (Figs. 2, 3 and 5). Third, pericytes and their processes were apparently more frequently observed and markedly larger as compared with those in C57BL mice. Actin-like microfilaments were abundant and exhibited localized assemblageinto conspicuous dense bodies, not only at the periphery but also at the center of the cytoplasm. As a result, altered pericytes resembled smooth muscle cells (Figs. 5 and 6 and Fig. 7). Fourth, the pericapillary space was frequently enlarged, and contained flocculous materials, collagen fibrils and fibroblasts, indicating leakage of plasma con-

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Fig. 4. Islet tissue of a dbjdb mouse aged 12 months. An intensely dilated capillary is seen. Wide pericapillary space contains numerous collagen fibrils (CF), fibroblasts (FB) and flocculous materials. A large pericyte process (PP) is seen. Endothelial cell (EC), erythrocyte (RBC). Fixed in modified Dalton’s fixative. x 4860

stituents (edema) and fibrosis (Figs. 2-4). Throughout this study, islet capillaries were less frequently observed in dbjdb than in age-matched C57BL mice. All the above abnormalities became more pronounced with age. Pericapillary edema and fibrosis were more frequently observed in islet periphery than in its center, but topographical predilection was not obvious for other abnormalities.

Contrary to our expectation, however, overall thickening of the basement membrane was hardly noticed in islet capillaries of db/db mice. Further, it must be added that such changes of the islet as B cell degranulation (Fig. 5), expansion of the endoplasmic reticulum and Golgi apparatus of B cells (Fig. 5), and occurrence of insular ductules (12 months age only) were visualized throughout our db/db mice, in accordance with a previous report [16].

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Fig. 5. Islet tissue of a dbjdb mouse aged 12 months. A portion of a capillary (CAP) is seen. The capillary is characterized by hypertrophied pericyte and irregular luminal contour. Pericyte processes(PP) contain an abundance of actin-like microfilaments and well-developed cell organelles. Dense bodies (arrow) due to assemblage of microfilaments are seen. B cells (B) are degranulated and contain well-developed endoplasmic reticulum. EC, endothelial cell; RBC, erythrocyte. Fixed in modified Dalton’s fixative. x 7000. Fig. 6. Portions of pericyte processesof an islet capillary from a 12-month C57BL mouse. The processescontain moderate amount of actin-like microfilaments (AM). These microfilaments are slightly clustered at the cytoplasmic periphery (arrow). M, mitochondria. Fixed in modified Dalton’s fixative. x 21200. Fig. 7. A portion of a pericyte process of an islet capillary from a 12-month dbjdb mouse. The process contain$ an abundance of actin-like microfilaments (AM). These microfilaments are assembled into conspicuous dense bodies (arrow). M, mitwhondria. Fixed in modified Dalton’s fixative. x 14000.

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Table I Parameters of islet capillary (mean k SD) Mouse, age

ICD (number/pm’)

C57 BL, 4 months, 0.0012 ) (n = 12) 63 0.0010 * db/db, 4 months, (n = 31) 53 C57 BL, 6 months, 0.0012 f (n = 26) 65 0.0007 + db/db, 6 months, (n = 26) $4 C57 BL, 12 months, 0.0012 k (n = 24) s4 0.0006 & db/db, 12 months, (n = 30) s4

0.0005

EEC (v)

LII

25.516 & 17.712

2.507 (n = 2.691 (n = 2.387

(n = 98)

0.0004

30.139 k 22.105 (n = 233)

0.0003 0.0001 0.0004 0.0002

28.231 & 16.553 (n = 214) 36.151 k 20.971 (n = 141) 28.027 k 14.716 (n = 206) 40.444 k 27.222 (n = 258)

+ 0.880 92) + 1.028 219) + 0.858

(n = 208)

2.821 k 1.132 (n = 138) 2.384 k 0.881

PAC (p2/capillary)

PAEEC ~rn’/~rn

2.546 + 4.321 (n = 96) 6.261 + 16.844

0.108 (n = 0.170 (n = 0.104

(n = 232)

2.959 + 5.395 (n = 214) 7.072 f 10.058 (n = 141) 3.505 * 8.314

k 0.192 96) + 0.306 232) + 0.189

(n = 214)

0.200 f 0.296 (n = 141) 0.119 If: 0.250

(n = 197)

(n = 200)

(n = 200)

2.652 f 1.197 (n = 252)

9.253 k 15.030

0.231 + 0.382

(n = 256)

(n = 256)

ICD, islet capillary density; EEC, external endothelial circumference; LII, lumal irregularity index; PAC, pericyte area per capillary; PAEEC, pericyte area per unit external endothelial circumference.Note: luminal irregularity index = internal endothelial circumference (pm)/2Jrr x luminal area (pm*)

3.3. Electron microscopic findings by morphometry

The following parameters were determined or calculated: islet capillary density (ICD), external endothelial circumference (EEC), luminal irregularity index (LII), pericyte area per capillary (PAC), and pericyte area per unit external endothelial circumference (PAEEC). The mean and SD. values of respective parameters are shown in Table 1, and P values for differences between strains and ages of the mouse given in Table 2. At 4 months of age, only EEC and PAC exhibited significant increase in db/db compared with C57BL mice. Other parameters also exhibited changes in the mean in dbjdb mice (decrease for ICD, increase for LII and PAEEC), but differences did not achieve statistical significance. At higher (6 and 12 months) ages, ICD was significantly decreased and other parameters were significantly increased in db/db compared with C57BL mice (Tables 1 and 2). In addition, age-dependent changes of some parameters were detected statistically in each strain. They are: decreaseof ICD at 6 and 12 months of age below the level at 4 months of age among db/db mice; increase of EEC at 6 and 12 months of age over

the level at 4 months of age among both strain mice; and increase of PAC and PAEEC (marginally significant at 12 versus 4 months) at 6 and 12 months of ages over the values at 4 months of age among db/db mice (Tables 1 and 2). As evident from Table 1, standard deviations of parameters other than ICD are consistently much greater in dbjdb than in C57BL mice. Although statistical significance cannot be tested due to limited numbers of animals, this result suggests exaggerated diversity of the size and shape of islet capillaries in dbjdb mice. 3.4. Preliminary

examination in other organs

The following organs were preliminarily examined electron microscopically: the cecum, retina, sciatic nerve and kidney. These organs were selected, because they have been shown to be susceptible to diabetes-associated changes in the function and morphology of the microvasculture. For the cecum, in particular, it has recently been reported that the capillary permeability is strikingly elevated in streptozotocin-diabetic rats [ 17,181,although the structural basis for the abnormality remains to be elucidated. As a result of the present examination, an apparent hypertrophy of the pericyte was noted in

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Table 2 P-values of parameters between groups Groupd compared

~4, d4 c6, d6 ~12, d12 ~4, c6 c4, cl2 c6, cl2 d4, d6 d4, d12 d6, d12

Parameters ICD

EEC

LII

PAC

PAEEC

0.13
0.04
0.16 10.01 0.02 0.22 0.20 0.93 0.32 0.35 0.08

0.01
0.10
Note; For ICD, comparisons are carried out without logarithmic transformation. For other parameters comparisons are carried out after logarithmic transformation. For animal groups, alphabetical letters and numerals respectively denote animal types and age in

the capillaries of dbjdb mice localized at: the tunica propria mucosae of the cecum (Fig. 8); the retinal nerve fiber layer (Fig. 9); and the endoneurium (Fig. 10). In the kidney of these diabetic mice, the mesangial cell, a cell type comparable to the pericyte was found to be provided with extremely profuse cytoplasmic processes(Fig. 11). 4. Discussion Diabetic microangiopathy of the pancreatic islet, if present, is of particular significance, since it could further aggravate diabetes mellitus, thus forming a vicious cycle. Nevertheless, only a few studies [2,11], have thus far been made on this theme, and this is probably the first study in which islet capillaries of spontaneously diabetic animals have been systematically examined by means of transmission electron microscopy. Wyse and Dulin [13] have reported on the age-related variation of clinical and biochemical parameters in dbjdb mice. Referring to their data, it can be estimated that normoinsulinemia and decreased pancreatic insulin content coexist with hyperglycemia in our dbjdb mice aged 4, 6 and 12 months. In the present study, a series of ultrastructural changes of islet capillaries were detected in dbjdb mice. They are: (1) capillary scarcity; (2) increase

in the mean and diversity of capillary size; (3) pericapillary edema and fibrosis; (4) hypertrophy of the pericyte and abundance therein of actin-like microfilaments; and (5) irregularity of the endothelial luminal surface. Some of these changes are commented on below. Changes (2) and (3) suggest that the islet capillary bed of dbjdb mice undergoes hyperperfusion, capillary hypertension and enhanced permeation of plasma constituents. This suggestion is consistent with lines of clinical and experimental evidence that blood flow is increased in some organs such as the retina and kidney in early diabetes in humans and animals [19-241, and that transcapillary passageof labeled molecule is accelerated in a series of diabetic organs and tissues [17,18,25-271. Groups of distinguished authors such as Parving et al. [23], Zatz and Brenner [24] and Williamson et al. [28] have speculated that the above changes of hemodynamics and capillary permeability in diabetes contribute to the formation of structural abnormalities of microvessels. Pericapillary proliferation of collagen fibrils, as observed in dbjdb mouse islets, can be taken as an adaptive mechanism for possible capillary hypertension. In this regard, it is interesting to mention that experimental hypertension in the rat has been reported to stimulate the production of collagen by arteries and cerebral microvessels [29,30]. Alternatively, it may be that enhanced cross-linking

M. Nakamura et al. 1 Diabetes Research and Clinical Practice 30 (1995) 89-100

Fig. 8. A portion of cecal tunica propria mucosae of a 6-month dbjdb mouse. A hypertrophied are !seen to surround a capillary (CAP). EC, endothelial cell. Perfusion-fixed with Karnovsky’s

pericyte (P) and its processes fixative. x 5940.

(PP)

Fig. 9. A portion of retinal nerve fiber layer of a 6-month dbjdb mouse. A hypertrophied pericyte (P) and its processes (PP) are to Sl.trround capillaries. EC, endothelial cell. Perfusion-fixed with Karnovcsky’s fixative. x 5940. Fig. 10. An endoneurial capillary of a 4-month db/db mouse. The capillary jthelial cell. Perfusion-fixed with Karnovsky’s fixative. x 5940.

is surrounded

by a hypertrophied

pericyte (P). EC

Fig. 11. A portion of a renal glomerulus of a 6-month dbjdb mouse. The mesangium is packed with mesangial cells (MC) and their prof use processes (MCP). Perfusion-fixed with Karnovsky’s fixative. x 3640.

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of collagen in diabetes [31-331 is responsible for the observed pericapillary fibrosis. Obviously, the most pivotal finding in this study is the hypertrophy of the pericyte and abundance therein of actin-like microfilaments observed in db/db mouse islets. Altered pericytes assumed resemblance to smooth muscle cells. Preliminary examination disclosed similar changes of the pericyte in organs with capillaries liable to be affected by diabetes, suggesting generalized occurrence of such pericytic changes and their relationship to diabetes. The contractility of the pericyte has been evidenced by recent histochemical and ultrastructural studies [34-371. Taken together, we are tempted to propose that the hypertrophic and hyperfunctional changes of the pericyte in db/db mice may represent a second adaptive mechanism for capillary hyperperfusion and capillary hypertension. Previous studies on two types of experimental hypertension in the rat appear to favor our consideration. First, retinal capillary pericytes have been shown to undergo hypertrophy and increase in actin filaments in response to experimental systemic hypertension [38]. Second, transformation of pericytes to smooth muscle cells has been visualized in the lung of the rat rendered pulmonary-hypertensive [39,40]. The above idea that the hypertrophic and hyperfunctional changes of the pericyte may result from altered hemodynamics in diabetes, however, remains speculative, since compelling evidence for it is not available. Hypertrophy of the pericyte or mesangial cell, as observed here in the islet and other organs of db/db mice, contrasts with a number of studies reporting loss or degeneration of the corresponding cells in the retina and skeletal muscles of diabetic patients and animals [3,41-431. Nevertheless, two groups of authors have provided results essentially similar to the present findings. Thus, Save-Sonderbergh et al. [44] have noticed the occurrence of the pericyte with a large, prominent and hyperchromatic nucleus in the subcutaneous tissue of diabetic patients, and Tilton et al. [45] have recently disclosed higher than normal proportions of capillary endothelial cell circumference covered by the pericyte in the retinas of streptozotocin-diabetic rats. It should be pointed out that our db/db mice as well as the rats studied

by Tilton et al. were diabetic for periods not exceeding 1 year, whereas diabetic subjects exhibiting reported loss or degeneration of the pericyte had been diabetic for years. In this context, it is also interesting to refer to the recent report [46] that proliferation of the mesangial cell precedes expansion of mesangial matrix in streptozotocin-diabetic rats. An important point yet to be discussed is that in the pancreatic islets of db/db mice overall thickening of the basement membrane was missing in the face of pericapillary fibrosis. Thickening of the basement membrane was also missing in other organs examined. This is apparently at variance with the reported prevalence in diabetes of capillary basement membrane thickening [1,3,511,47-491 and concurring basement membraneassociated pericapillary fibrosis [47-491. The reason for the above discrepancy between the present and previous studies cannot be explained unequivocally at present. Conceivably, factors such as the pathogenesis and duration of diabetes and the species of animals may be responsible. Finally, it is possible that the above changes, such as pericapillary edema and fibrosis and pericyte hypertrophy, acting in concert, compromise the transfer of gasses, nutrients and hormones between the capillary and islet cells, and thereby badly affect insular hormone secretion and the extent of the diabetic state.

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