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Disruption of blood-retinal barrier in experimental diabetic rats: An electron microscopic study
hp.
Bye Res. (1980) 30,401-410
Disruption of Blood-Retinal Barrier in Experimental Diabetic Rats: An Electron Microscopic Study TATSURO
ISHIBASHI, KENZO
TANAKA AND YOSHIAKI
TANIGUCHI
Depa,rtnzent of Pathology and Ophthalmology, Faculty of Medicine, Kyushu University. 3-l-l Maidashi, Hignshi-ku Fukuoka 812, Jnpmf, (Received 7 Mary 1979 and in revised form 8 Sqxkmber
19?‘9, NW
York)
Permeability pattern of retinal capillaries in streptozotocin-diabetic rats, maintained for l--6 months, was studied with the electron microscope, using horseradish peroxidase as a tracer. A reaction product of horseradish peroxidase was confined to the capillary lumina and to a small number of vesicles on the luminal portion of the endothelial cells in control rst,s and diabetic rats maintained for 1 month. In diabetic rats maintained for 2-6 months. reaction product was observed in the basement membrane of endothelial cells and pericytes and extended to the extracellular spaces around the capillaries. The number of reaction product-labeled vesicles in the capillary endothelial cells was greatly increased in diabetic rats maintained for 2 months, as compared with that in control rats and diabetic rats maintained for 1 month. Reaction product was also found in the intercellular junctions of capillary endothelisl cells from the luminal to the abluminal portion in diabetic rats maint.ained for :S-6 months. The increased permeability in retinal capillaries of diabetic rats preceded the thickening of the basement membrane, and seemed t,o play an important role in the development of diabetic retinopathy. retinal capillaries; vesicular transport; inter.h’e!l rcords : st,reptozotocin diabetes; cellular junct,ions; blood-retinal barrier; permeability.
1. Introduction Thickening of capillary basement membrane has been considered a typical feature of diabetic retinopathy by several authors (Bloodworth and Molitor, 1965; Taniguchi and Nomura, 1968; Leuenberger, Cameron, Stauffacher, Renold and Babel, 1971: Babel and Leuenberger, 19’74; Papachristodoulou and Heath, 1977). In recent years, a breakdown of the blood-retinal barrier to fluorescein has been reported as the earliest measurable change in the retinal vessels in human diabetes mellitus (CunhaVaz, De Abreu? Campos and Pigo, 1975) and in streptozotocin-diabetic rats (Waltman, Krupin. Hanish, Oestrich and Becker, 1978). It has been speculated that the enhanced permeability plays an important role in the pathogenesis of diabetic retinopathy, but the early disruption of the blood-retinal barrier has not yet been reportecl at the ultrastructural level. To elucidate the pathogenesis of diabetic retinopathy with reference to the changes of the permeability pattern, the retinal capillaries of streptozotocin-diabetic rats. maintained for l-6 months, were studied with the electron microscope. The permeal)ilit.y was st.udicd by the horseradish peroxidase t,echnique. 2. Materials and Methods Twenty-four male Wistar-King A rats weighing approximately 200 g were injected intravenously with 65 mg streptozotocin/kg body weight, dissolved in 0.3 ml of citrate buffer (pH 4.5) (diabetic group). Four control rats of the same body weight were injected intravenously
with a corresponding
0014-4836/80/040401$-10
volulne
of a solution
of O+q/, NaCl (control
group).
0 1980 Academic Press Inc. (London) Limited
$01.00/0 401
402
T. ZSHIKASHI.
K. TANAKA
ANl)
Y. ‘I‘ABI(:U(!HI
The animals were regularly assessed(every 3 weeks) for weight, blood glucose wii(l pl!-c.~jsuria. The blood glucose was checked with Dextrostix and glycosuria with Tes-Tape. Diabetes was established by persistent hyperglycemia (> 300 mp/lO() ml). glycAosuri;r. polyuria and impaired growth. Preparution
of tissues
Twenty-four diabetic rats were killed at periods varying l-6 months after the injection of streptozotocin and divided into three groups; (1) three rats maintained for 1 month. (2) seven rats for 2 months, (3) 14 rats for 3-6 months. Four control rats were killed 6 months after the injection of the control solution. These rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (9 mg/lOO g body weight). Fifteen to 30 min. before killing, the tail vein of each rat was injected with 20 mg horseradish peroxidase (HRP) (Sigma Chemical Co., Type II)/100 g body weight, dissolved in 0.5 ml of isotonic saline, except for one control rat examined on the endogenous peroxidase activity. The eyes were enucleated and fixed with 3% glutaraldehyde buffered with 0.1 M-cacodylate for 3 hr at 4°C. The tissue was washed overnight at 4°C in 0.1 M-cacodylate buffer and then sectioned at a thickness of 100 pm. The specimens were incubated for 15 min at room temperature in 10 ml of 0.05 iv-Tris-HCl buffer (pH ‘76), containing O.Ol”/O hydrogen peroxide and 5 mg of 3,3’-diaminobenzidine tetrahydrochloride (Merck, Germany). After post-fixation with 1% OsO,, buffered with O-1 M-cacodylate for 1 hr, the specimens were dehydrated in graded ethanol, treated with propylene oxide, and embedded in Epon 812. Thin sections were cut on a LKB ultratome, stained with lead citrate. These were then examined with a JEM 1OOCelectron microscope. Morphometric
analysis
of oesicular transport in the elzdothelial
cells
For morphometric analysis of vesicular transport in the endothelial cells, tissue blocks were selected from each of the two rats in the control group and in the diabetic groups maintained for 1 month and for 2 months. Twenty capillaries were randomly selected from the retinal outer plexiform layer in each group. The electron micrographs containing a capillary were originally taken at magnification of x 6700 or x 10 000 in order to assess the capillary’s inner diameter. Two or more micrographs of the endothelial cells in each capillary were taken at an original magnification of x20 000, and then enlarged to a final magnification of x50 000. Morphometry was performed on the peripheral zone of each capillary according to Simionescu, Simionescu and Palade (1974). Measurement of diameter was made on the micrographs with a vernier caliper, and that of area with a Zero Setting Roller Planimeter (Uchidayoko, Tokyo, Japan). The following data were measured or counted: irmer diameter (pm), number of vesicles (/pm2), labeling rate for vesicles with reaction product (94). 3. Results The biochemical data characterizing were summarized in Table I. Permeability
of retinal
capillaries
the metabolic state of the rats under study
to HRP
Control rats. In three control rats, the reaction product of HRP was confined to the capillary lumina and to a small number of vesicles on the luminal portion of the endothelial cells. These vesicles were measured about 400-700 A in diameter, although larger ones occurred, probably representing fusions of the smaller vesicles. Reaction product was not observed either in the intercellular junctions of the endothelial cells or in the basement membrane. Extracellular spaces of the glial cells and the neural cells around the capillaries were also devoid of reaction product (Fig. 1).
BLOOD-RETINAL
BARRIER TABLE
IN
DIABETES
403
I
Biochemical data Blood glucose (mg/lOO ml)
Glycosuria,
Urine volume (/24 hr, ml)
Body weight (8)
Control group*
106.3*
(n = 4) Diabetic groups 1 month
410.0f20+3
t+-t+)
95.0&12.1
206.0&
4.2
425.7* 16.0
(+++I
97.1&
9.2
220.01
7.1
432.9f15.7
c+++j
112.3*
8.8
231.6% 4.1
2 rn!ZG 3, (n = 7) 3-6 months (72.= 14)
5.2
(-)
1.31
0.2
a = number of rats The values represent meanfs.E. * Rats in the control group were killed 6 months after intravenous control solution.
405.3*10.2
injection
of the
In one control rat examined on the endogenous peroxidase activity, reaction product was observed within the lysosomes and the multivesicular bodies in the endothelial cells and pericytes, but vesicles, basement membrane, and the intercellular junctions of the endothelial cells were free from reaction product. Diabetic rats maintained for 1 month. In three diabetic rats, thickening of the capillary basement membrane was not observed. The rate of labeling with reaction product for vesicles in the endothelial cells was almost the same as that in control rats. The intercellular junctions of the endothelial cells were devoid of reaction product (Fig. 2). Diabetic rats maintained for 2 months. In seven diabetic rats, no thickening of the capillary basement membrane was observed. Endothelial cells contained a large number of vesicles with reaction product (Fig. 3). Reaction product was present within the luminal portion of the intercellular junctions, but was absent beyond the first fusion point between the outer leaflets of the plasma membrane. Diabetic rats rna~nta~n~ for 3-6 months. In 14 diabetic rats, focal thickening of the capillary basement membrane was observed, but endothelial cells and pericytes showed no degenerative changes. In capillaries showing no thickening of the basement membrane, a large number of reaction product-labeled vesicles were found in the capillary endothelial cells. Some labeled vesicles on the abluminal portion of the endothelial cells opened into the basement membrane and discharged HRP into it (Figs 4, 5). However, no reaction product penetrated the capillary endothelial cells. from the capillary lumen to the basement membrane, via transendothelial channels. Some pericytes contained various sized vesicles with reaction product (Fig. 5). Dense deposits of reaction product were present in the basement membrane of the endothelial cells (Figs 4, 5) and of the pericytes (Fig. 5).
Brc. 1. Retinal capillary of control rat (15 min after injection of HRI’). Reaction product is found ill the lumen and in a small number of vesicles, but neither in the intercellular junctions of the w(hlthrlial cells (arrows) nor in the basement membrane (BM). R. red blood cell ( “’ 15 300).
FIG. 2. Retinal capillary of diabetic rat maintained for 1 month (30 min after injection of HKP). Reaction product is found in the lumen and in a small number of vesicles, but neither in the intcrcellular junction of the endothelial cells (arrow) nor in the basement membrane (BM). Thickening of t,he basement membrane (BM) is not found. R, red blood cell ( x 15 500).
BLOOD-RETINAL
BARRIER
IN
DIABETES
405
Pm. 3. Retinal capillary of diabetic rat maintained for 2 months (15 min after injection of HRP). The endothelial cells contain a large number of vesicles labeled with reaction product. Thickening of thv basement membrane (BM) is not found ( ;~:23 000).
FIG. 4. Retinal capillary of diabetic rat maintained for 3 months (15 min after injection of HRP). The endothelial cell contains a large number of reaction product-labeled vesicles which open into the basement membrane (BM). The basement membrane (BM) of the endothelial cell is filled with reaction product, but the competent intercellular junction impedes the transit of HRP (arrow). L, lumen of capillary ( x 74 000).
FIG. 5. Retinal capillary of diabetic I,at maintained for 3 months (30 mill after injrc:t ioll (,t‘ H 1%~). Reaction product-containing vesicles open into the basement membrane (l3M). ‘I’hc basen~nt ~ncmb~~anr (EM) of the endothelial cell and pericyte is filled wit’h reaction product,. The vesicles in thr- per~i+~~ (t’) arc also filled with reaction product. L. lumen of capillary R, red blood cell ( 59 000).
FIG. 6. Retinal capillary of diabetic rat maintained for 3 months (15 min after injection of HRP). The intercellular junction of the endothelial cells (arrow) is filled with reaction product. Reaction product in the basement membrane shows higher density in the opening site of the intercellular junction. The basement membrane is not thickened. R., red blood cell ( b. I1 800).
BLOOD-RETINAL
BARRIER
In- DIABETES
407
FIG. 7. Higher magnification of the intercellular junction of the endothelial cells (15 min after inof HRP). The intercellular junction of the endothelial cells (arrow) is filled with reaction product). (‘lose apposition of the membrane is found in two points within the intercellular junction ( ..I 44 000). jection
FIG. 8. Retina of diabetic rat maintained for 3 mouths (30 min after inject.ion of HRP). The basement membrane of capillary endothelial cells (BM) is filled with reaction product. Reaction product is also found in the extracellular spaces of the retinal cells. L, lumen of capillary ( Y 20 000).
40s
‘1’. ISHIBSSHI.
K. TANAKA
XXD
1’. TASL(:
[.(‘I-1 I
Some intercellular junctions of the endothelial cells were filled wit,h reaction product. Reaction product in the basement membrane showed higher density in t,htl areas where the intercellular junctions opened into the basement membrane. The basement membrane containing reaction product was not thickened (Fig. 6). ilit higher magnification, the intercellular junctions showed close apposition of the membrane in two or three points. But density of reaction product within the intercellular junctions was the same from the luminal to the abluminal portion (Fig. 7). Reaction product was consistently found in the extracellular spaces of the glial cells and the neural cells around the capillaries. Diffuse uptake of HRP int#o the cytoplasm of glial cells and neural cells was not observed (Fig. 8).
The inner diameter of capillaries used for morphometry was similar in each of the experimental groups. The number of vesicles (/qlz) was 14.4k1.5 (mean&S.E.) in control group, 14*9&1*7 in diabetic group maintained for 1 month and 33+3&l-2 for 2 months. The labeling rate for vesicles with reaction product (9,;) was 15*0*1.0 (1lleall~S.E.) in the control group, 15.3&l -2 in the diabetic group maintained for 1 mont)h and 37.2*1.3 for 2 months. TABLE II
3forphometric ar~alysis of ,vesicular traxyort
Capillary diameter (pm) Number of vesicles (/pm”) Labeling rate for vesicles with reaction product (yo)
n = The *P t P
ill the mdotllelial cells
C’ontrol group
Diabetic group (1 month)
Diabetic group (3 months)
(n = 20) 3.4*0.1 14.4+1.5
(n = 20) 3.5hO.l 14.9+ 1.7
(n = 20) 3.5,tO.l 33+?*1.2*
15.0&1.0
153+ 1.2
37.2,t1.3?
number of capillaries examined for the analysis values represent meanis.~. < 0.005 [vs. Control group, Diabetic group (1 month)] < 0.005 [vs. Control group, Diabetic group (1 month)]
The number of vesicles and the labeling rate for vesicles with reaction product in the diabetic group maintained for 2 months were significantly higher than those in the control group and in the diabetic group maintained for 1 month. 4. Discussion The concept of the blood-retinal barrier, analogous to the blood-brain barrier, was established by Cunha-Vaz, Shakib and Ashton (1966), using trypan blue and colloidal carbon as tracers. HRP (40 000 mol. wt and 60 A d’ram.) was introduced as a tracer for studying the permeability of various cells or tissues at an ultrastructural level (Graham and Karnovsky, 1966). Under normal conditions of the retinal vasculature, no reaction product of HRP
BLOOD-RETINAL
BARRIER
IK
DIABETES
409
was found within the intercellular junctions of the endothelial cells, and vesicular transport across the endothelial cell cytoplasm was negligible (Shiose, 1970). Rakieten, Rakieten and Nadkarni (1963) reported that streptozotocin, an antibiotic extracted from Streptomyces achromogenes, when administered intravenously led to frank diabetes in dogs and rats, due to a damaging the pancreatic beta cells. There were several reports concerning the retinal vessels of streptozotocin-diabetic rats (Leuenberger, Cameron, Stauffacher, Renold and Babel, 1971; Sosula, Beaumont, H0110ws and Jonson, 1972; Babel and Leuenberger, 1974; Papachristodoulou and Heath, 1977). But there were only a few reports about changes in t’he permeability pattern (Wallow and Engerman, 1977; Leuenberger and Babel, 1978). Wallow and Engerman (1977) reported an increased permeability of certain retinal vessels to HRP in longstanding, intentionally poorly controlled, alloxan-diabetic dogs. The) suggested that this increased permeability was due to a junctional insufficiency. However, these vascular damages seem to be considerably more severe than the ones descriljed in this paper. In t,he present study, the vesicular transport in diabetic rats maintained for 2-6 months was greatly increased as compared with that in control rats and diabetic rats maintained for 1 month, Junctional transport of HRP between the capillary endothelial cells was occasionally found in diabetic rats maint,ained for 3-6 months. HRP seemed to reach the basement membrane through broken tight junct,ions. However, it was not clear which route-vesicular or junctional transport-predominantly contributed to the increased permeability. According to the freeze-fracture image of the strands in endothelial junctions of muscle capillaries, the strands were discontinuous in part (Simionescu, Simionescu and Palade, 1975; Wissig and Williams, 1978). It could be speculated that HRP passes between an interruption in the network of strands and transfers to the basement membrane. Reaction product was not limited to the basement membrane or to the extracellular spaces of the glial cells. This finding shows that the endothelial cells and the intercellular junction of endothelial cells play an important role in the barrier function, compared with the surrounding basement membrane and the glial tissue. In capillaries showing no thickening of the basement membrane an increased permeability was found, so this increase in permeability of the endothelial layer preceded the thickening of the basement membrane. Our experiment,s have not ruled out the possibility of a specific effect of streptozotocin. Pretreatment with 2-deoxyglucose or 3-o-methylglucose prior to streptozotocin injection (Ganda, Rossini and Like, 1976) seems to be a better control than just saline injection. But no increased permeability was found in the diabetic rats maintained for 1 month in the present study. Direct. toxicity of streptozotocin could not be the cause of an increased vascular permeability in the diabetic ra,ts maintained for 2-6 months. It is known that HRP induces an increased vascular permeability when injected locally or systemat#ically in rats (Cotran and Karnovskp, 1967 ; Cotran and Karnovsky, of 1968). According to our unpublished observations, intravenous administration 20 mg/lOO g of HRP did not seem to induce an increased vascular permeability in Wistar-King A strain ra,ts. Based on the present experimental results, it seems reasonable to postulate that disruption of the blood-retinal barrier in retinal capillaries of streptozotocin-diabetic rats is due to an increase in vesicular transport and a junctional insufficiency hehween
410
T.lSHIHASHI,K.TANAKA
AXI) Y.'I'ASl(;L'~'HI
the endothelial cells. The increase in capillary permeabilit’y precedes the thickening of the basement membrane and may play an important role in the development ot diabet,ic retinopathy. REFERENCES Babel, J. and Lcuenberger, P. (1974). A long term study on the ocular lesions in streptozotocitl diabetic rats. Albrecht v. Graefes Arch. klin. exp. Ophthal. 189, 191-209. Bloodworth, J. M. B. and Molitor, D. L. (1965). Ultrastructural aspects of human and canine diabetic retinopathy. Invest. Ophthalmol. 4, 103748. Cotran, R. S. and Karnovsky, M. J. (1967). Vascular leakage induced by horseradish perosidase in the rat. Proc. Sot. Exp. Biol. Med. 126, 557-61. Cotmn, R. S. and Karnovsky, M. J. (1968). Resistance of Wistar/Furth rats to the mast celldamaging effect of horseradish peroxidase. J. Histochem. Cytochem. 16, 382-3. Cunha-Vaz, J. G., Shakib, M. and Ashton, N. (1966). Studies on the permeability of the bloodretinal barrier. I. On the existence, development, and site of a blood-retinal barrier. Br. J. Ophthalmol. 50, 441-53. Cunha-Vaz, J., De Abreu, J. R. F., Campos, A. J. and Figo, G. M. (1975). Early breakdown of the blood-retinal barrier in diabetes. Br. J. Ophthdmol. 59, 649-56. Ganda, 0. P., Rossini, A. A. and Like, A. A. (1976). St.udies on streptozotocin diabetes. Diabetes. 25,595603. Graham, R. C. and Karnovsky, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302. Leuenberger, P., Cameron, D., Stauffacher, W., Renold, A. E. and Babel, J. (1971). Ocular lesions in rats rendered chronically diabet,ic with streptozotocin. Ophthnl. Res. 2, 189-204. Leuenberger, P. M. and Babel, J. (1978). Retinal microangiopathy in streptozotocin diabetic rats. Cellular and biochemical aspects in diabetic retinopat,hy. INSERM Syw~posium. 7, 41-56. Papachristodoulou, D. and Heath, H. (1977). Ultrastructural alterations during the development of retinopathy in sucrose-fed and streptozotocin-diabetic rats. Exp. Eye Res. 25,371-84. Rakieten, N., Rakieten, M. L. and Nadkarni. RI. (1963). Studies on the diabetogenic action of streptozotocin. Cancer Chewlother. Rep. 29, 91-8. Shiose, Y. (1970). Electron microscopic studies on blood-retinal and blood-aqueous barriers. Jap. J. Ophthalmol. 14, 73-87. Simionescu, M., Simionescu, N. and Palade, G. E. (1974). Morphometric data on the endot,helium of blood capillaries. J. Cell Biol. 60,128-52. Simionescu. M., Simionescu, N. and Palade. G. E. (1975). Segmental differentiations of cell junctions in the vascular endothelium. J. Cell BioZ. 67, 863-85. Sosula, L.. Beaumont, P., Hollows, F. C. and Jonson, K. M. (1972). Dilatation and endothelial proliferation of retinal capillaries in streptozotocin-diabetic rats: Quantitative elect,ron microscopy. Invest. Ophthalmol. 11, 926-35. Taniguchi, Y. and Nomura. T. (1968). Fine structure of retinal blood vessels in human diabetics. Acta Sot. Ophthalmol. Jap. 72, 1165-78. Wallow, I. H. L. and Engerman, R. L. (1977). Permeability and patency of retinal blood vessels in experimental diabetes. Invest. OphthaZmoZ. 16, 447-61. Waltman, S., Krupin, T., Hanish, S., Oestrich, C. and Becker, B. (1978). Alteration of the bloodretinal barrier in experimental diabetes mellitus. Arch. Ophthdmol. 96,878-g. Wissig, S. L. and Williams, M. C. (1978). Permeability of muscle capillaries to microperoxidase. J. Cell Biol. 76, 341-59.
Bye Res. (1980) 30,401-410
Disruption of Blood-Retinal Barrier in Experimental Diabetic Rats: An Electron Microscopic Study TATSURO
ISHIBASHI, KENZO
TANAKA AND YOSHIAKI
TANIGUCHI
Depa,rtnzent of Pathology and Ophthalmology, Faculty of Medicine, Kyushu University. 3-l-l Maidashi, Hignshi-ku Fukuoka 812, Jnpmf, (Received 7 Mary 1979 and in revised form 8 Sqxkmber
19?‘9, NW
York)
Permeability pattern of retinal capillaries in streptozotocin-diabetic rats, maintained for l--6 months, was studied with the electron microscope, using horseradish peroxidase as a tracer. A reaction product of horseradish peroxidase was confined to the capillary lumina and to a small number of vesicles on the luminal portion of the endothelial cells in control rst,s and diabetic rats maintained for 1 month. In diabetic rats maintained for 2-6 months. reaction product was observed in the basement membrane of endothelial cells and pericytes and extended to the extracellular spaces around the capillaries. The number of reaction product-labeled vesicles in the capillary endothelial cells was greatly increased in diabetic rats maintained for 2 months, as compared with that in control rats and diabetic rats maintained for 1 month. Reaction product was also found in the intercellular junctions of capillary endothelisl cells from the luminal to the abluminal portion in diabetic rats maint.ained for :S-6 months. The increased permeability in retinal capillaries of diabetic rats preceded the thickening of the basement membrane, and seemed t,o play an important role in the development of diabetic retinopathy. retinal capillaries; vesicular transport; inter.h’e!l rcords : st,reptozotocin diabetes; cellular junct,ions; blood-retinal barrier; permeability.
1. Introduction Thickening of capillary basement membrane has been considered a typical feature of diabetic retinopathy by several authors (Bloodworth and Molitor, 1965; Taniguchi and Nomura, 1968; Leuenberger, Cameron, Stauffacher, Renold and Babel, 1971: Babel and Leuenberger, 19’74; Papachristodoulou and Heath, 1977). In recent years, a breakdown of the blood-retinal barrier to fluorescein has been reported as the earliest measurable change in the retinal vessels in human diabetes mellitus (CunhaVaz, De Abreu? Campos and Pigo, 1975) and in streptozotocin-diabetic rats (Waltman, Krupin. Hanish, Oestrich and Becker, 1978). It has been speculated that the enhanced permeability plays an important role in the pathogenesis of diabetic retinopathy, but the early disruption of the blood-retinal barrier has not yet been reportecl at the ultrastructural level. To elucidate the pathogenesis of diabetic retinopathy with reference to the changes of the permeability pattern, the retinal capillaries of streptozotocin-diabetic rats. maintained for l-6 months, were studied with the electron microscope. The permeal)ilit.y was st.udicd by the horseradish peroxidase t,echnique. 2. Materials and Methods Twenty-four male Wistar-King A rats weighing approximately 200 g were injected intravenously with 65 mg streptozotocin/kg body weight, dissolved in 0.3 ml of citrate buffer (pH 4.5) (diabetic group). Four control rats of the same body weight were injected intravenously
with a corresponding
0014-4836/80/040401$-10
volulne
of a solution
of O+q/, NaCl (control
group).
0 1980 Academic Press Inc. (London) Limited
$01.00/0 401
402
T. ZSHIKASHI.
K. TANAKA
ANl)
Y. ‘I‘ABI(:U(!HI
The animals were regularly assessed(every 3 weeks) for weight, blood glucose wii(l pl!-c.~jsuria. The blood glucose was checked with Dextrostix and glycosuria with Tes-Tape. Diabetes was established by persistent hyperglycemia (> 300 mp/lO() ml). glycAosuri;r. polyuria and impaired growth. Preparution
of tissues
Twenty-four diabetic rats were killed at periods varying l-6 months after the injection of streptozotocin and divided into three groups; (1) three rats maintained for 1 month. (2) seven rats for 2 months, (3) 14 rats for 3-6 months. Four control rats were killed 6 months after the injection of the control solution. These rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (9 mg/lOO g body weight). Fifteen to 30 min. before killing, the tail vein of each rat was injected with 20 mg horseradish peroxidase (HRP) (Sigma Chemical Co., Type II)/100 g body weight, dissolved in 0.5 ml of isotonic saline, except for one control rat examined on the endogenous peroxidase activity. The eyes were enucleated and fixed with 3% glutaraldehyde buffered with 0.1 M-cacodylate for 3 hr at 4°C. The tissue was washed overnight at 4°C in 0.1 M-cacodylate buffer and then sectioned at a thickness of 100 pm. The specimens were incubated for 15 min at room temperature in 10 ml of 0.05 iv-Tris-HCl buffer (pH ‘76), containing O.Ol”/O hydrogen peroxide and 5 mg of 3,3’-diaminobenzidine tetrahydrochloride (Merck, Germany). After post-fixation with 1% OsO,, buffered with O-1 M-cacodylate for 1 hr, the specimens were dehydrated in graded ethanol, treated with propylene oxide, and embedded in Epon 812. Thin sections were cut on a LKB ultratome, stained with lead citrate. These were then examined with a JEM 1OOCelectron microscope. Morphometric
analysis
of oesicular transport in the elzdothelial
cells
For morphometric analysis of vesicular transport in the endothelial cells, tissue blocks were selected from each of the two rats in the control group and in the diabetic groups maintained for 1 month and for 2 months. Twenty capillaries were randomly selected from the retinal outer plexiform layer in each group. The electron micrographs containing a capillary were originally taken at magnification of x 6700 or x 10 000 in order to assess the capillary’s inner diameter. Two or more micrographs of the endothelial cells in each capillary were taken at an original magnification of x20 000, and then enlarged to a final magnification of x50 000. Morphometry was performed on the peripheral zone of each capillary according to Simionescu, Simionescu and Palade (1974). Measurement of diameter was made on the micrographs with a vernier caliper, and that of area with a Zero Setting Roller Planimeter (Uchidayoko, Tokyo, Japan). The following data were measured or counted: irmer diameter (pm), number of vesicles (/pm2), labeling rate for vesicles with reaction product (94). 3. Results The biochemical data characterizing were summarized in Table I. Permeability
of retinal
capillaries
the metabolic state of the rats under study
to HRP
Control rats. In three control rats, the reaction product of HRP was confined to the capillary lumina and to a small number of vesicles on the luminal portion of the endothelial cells. These vesicles were measured about 400-700 A in diameter, although larger ones occurred, probably representing fusions of the smaller vesicles. Reaction product was not observed either in the intercellular junctions of the endothelial cells or in the basement membrane. Extracellular spaces of the glial cells and the neural cells around the capillaries were also devoid of reaction product (Fig. 1).
BLOOD-RETINAL
BARRIER TABLE
IN
DIABETES
403
I
Biochemical data Blood glucose (mg/lOO ml)
Glycosuria,
Urine volume (/24 hr, ml)
Body weight (8)
Control group*
106.3*
(n = 4) Diabetic groups 1 month
410.0f20+3
t+-t+)
95.0&12.1
206.0&
4.2
425.7* 16.0
(+++I
97.1&
9.2
220.01
7.1
432.9f15.7
c+++j
112.3*
8.8
231.6% 4.1
2 rn!ZG 3, (n = 7) 3-6 months (72.= 14)
5.2
(-)
1.31
0.2
a = number of rats The values represent meanfs.E. * Rats in the control group were killed 6 months after intravenous control solution.
405.3*10.2
injection
of the
In one control rat examined on the endogenous peroxidase activity, reaction product was observed within the lysosomes and the multivesicular bodies in the endothelial cells and pericytes, but vesicles, basement membrane, and the intercellular junctions of the endothelial cells were free from reaction product. Diabetic rats maintained for 1 month. In three diabetic rats, thickening of the capillary basement membrane was not observed. The rate of labeling with reaction product for vesicles in the endothelial cells was almost the same as that in control rats. The intercellular junctions of the endothelial cells were devoid of reaction product (Fig. 2). Diabetic rats maintained for 2 months. In seven diabetic rats, no thickening of the capillary basement membrane was observed. Endothelial cells contained a large number of vesicles with reaction product (Fig. 3). Reaction product was present within the luminal portion of the intercellular junctions, but was absent beyond the first fusion point between the outer leaflets of the plasma membrane. Diabetic rats rna~nta~n~ for 3-6 months. In 14 diabetic rats, focal thickening of the capillary basement membrane was observed, but endothelial cells and pericytes showed no degenerative changes. In capillaries showing no thickening of the basement membrane, a large number of reaction product-labeled vesicles were found in the capillary endothelial cells. Some labeled vesicles on the abluminal portion of the endothelial cells opened into the basement membrane and discharged HRP into it (Figs 4, 5). However, no reaction product penetrated the capillary endothelial cells. from the capillary lumen to the basement membrane, via transendothelial channels. Some pericytes contained various sized vesicles with reaction product (Fig. 5). Dense deposits of reaction product were present in the basement membrane of the endothelial cells (Figs 4, 5) and of the pericytes (Fig. 5).
Brc. 1. Retinal capillary of control rat (15 min after injection of HRI’). Reaction product is found ill the lumen and in a small number of vesicles, but neither in the intercellular junctions of the w(hlthrlial cells (arrows) nor in the basement membrane (BM). R. red blood cell ( “’ 15 300).
FIG. 2. Retinal capillary of diabetic rat maintained for 1 month (30 min after injection of HKP). Reaction product is found in the lumen and in a small number of vesicles, but neither in the intcrcellular junction of the endothelial cells (arrow) nor in the basement membrane (BM). Thickening of t,he basement membrane (BM) is not found. R, red blood cell ( x 15 500).
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Pm. 3. Retinal capillary of diabetic rat maintained for 2 months (15 min after injection of HRP). The endothelial cells contain a large number of vesicles labeled with reaction product. Thickening of thv basement membrane (BM) is not found ( ;~:23 000).
FIG. 4. Retinal capillary of diabetic rat maintained for 3 months (15 min after injection of HRP). The endothelial cell contains a large number of reaction product-labeled vesicles which open into the basement membrane (BM). The basement membrane (BM) of the endothelial cell is filled with reaction product, but the competent intercellular junction impedes the transit of HRP (arrow). L, lumen of capillary ( x 74 000).
FIG. 5. Retinal capillary of diabetic I,at maintained for 3 months (30 mill after injrc:t ioll (,t‘ H 1%~). Reaction product-containing vesicles open into the basement membrane (l3M). ‘I’hc basen~nt ~ncmb~~anr (EM) of the endothelial cell and pericyte is filled wit’h reaction product,. The vesicles in thr- per~i+~~ (t’) arc also filled with reaction product. L. lumen of capillary R, red blood cell ( 59 000).
FIG. 6. Retinal capillary of diabetic rat maintained for 3 months (15 min after injection of HRP). The intercellular junction of the endothelial cells (arrow) is filled with reaction product. Reaction product in the basement membrane shows higher density in the opening site of the intercellular junction. The basement membrane is not thickened. R., red blood cell ( b. I1 800).
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FIG. 7. Higher magnification of the intercellular junction of the endothelial cells (15 min after inof HRP). The intercellular junction of the endothelial cells (arrow) is filled with reaction product). (‘lose apposition of the membrane is found in two points within the intercellular junction ( ..I 44 000). jection
FIG. 8. Retina of diabetic rat maintained for 3 mouths (30 min after inject.ion of HRP). The basement membrane of capillary endothelial cells (BM) is filled with reaction product. Reaction product is also found in the extracellular spaces of the retinal cells. L, lumen of capillary ( Y 20 000).
40s
‘1’. ISHIBSSHI.
K. TANAKA
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Some intercellular junctions of the endothelial cells were filled wit,h reaction product. Reaction product in the basement membrane showed higher density in t,htl areas where the intercellular junctions opened into the basement membrane. The basement membrane containing reaction product was not thickened (Fig. 6). ilit higher magnification, the intercellular junctions showed close apposition of the membrane in two or three points. But density of reaction product within the intercellular junctions was the same from the luminal to the abluminal portion (Fig. 7). Reaction product was consistently found in the extracellular spaces of the glial cells and the neural cells around the capillaries. Diffuse uptake of HRP int#o the cytoplasm of glial cells and neural cells was not observed (Fig. 8).
The inner diameter of capillaries used for morphometry was similar in each of the experimental groups. The number of vesicles (/qlz) was 14.4k1.5 (mean&S.E.) in control group, 14*9&1*7 in diabetic group maintained for 1 month and 33+3&l-2 for 2 months. The labeling rate for vesicles with reaction product (9,;) was 15*0*1.0 (1lleall~S.E.) in the control group, 15.3&l -2 in the diabetic group maintained for 1 mont)h and 37.2*1.3 for 2 months. TABLE II
3forphometric ar~alysis of ,vesicular traxyort
Capillary diameter (pm) Number of vesicles (/pm”) Labeling rate for vesicles with reaction product (yo)
n = The *P t P
ill the mdotllelial cells
C’ontrol group
Diabetic group (1 month)
Diabetic group (3 months)
(n = 20) 3.4*0.1 14.4+1.5
(n = 20) 3.5hO.l 14.9+ 1.7
(n = 20) 3.5,tO.l 33+?*1.2*
15.0&1.0
153+ 1.2
37.2,t1.3?
number of capillaries examined for the analysis values represent meanis.~. < 0.005 [vs. Control group, Diabetic group (1 month)] < 0.005 [vs. Control group, Diabetic group (1 month)]
The number of vesicles and the labeling rate for vesicles with reaction product in the diabetic group maintained for 2 months were significantly higher than those in the control group and in the diabetic group maintained for 1 month. 4. Discussion The concept of the blood-retinal barrier, analogous to the blood-brain barrier, was established by Cunha-Vaz, Shakib and Ashton (1966), using trypan blue and colloidal carbon as tracers. HRP (40 000 mol. wt and 60 A d’ram.) was introduced as a tracer for studying the permeability of various cells or tissues at an ultrastructural level (Graham and Karnovsky, 1966). Under normal conditions of the retinal vasculature, no reaction product of HRP
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was found within the intercellular junctions of the endothelial cells, and vesicular transport across the endothelial cell cytoplasm was negligible (Shiose, 1970). Rakieten, Rakieten and Nadkarni (1963) reported that streptozotocin, an antibiotic extracted from Streptomyces achromogenes, when administered intravenously led to frank diabetes in dogs and rats, due to a damaging the pancreatic beta cells. There were several reports concerning the retinal vessels of streptozotocin-diabetic rats (Leuenberger, Cameron, Stauffacher, Renold and Babel, 1971; Sosula, Beaumont, H0110ws and Jonson, 1972; Babel and Leuenberger, 1974; Papachristodoulou and Heath, 1977). But there were only a few reports about changes in t’he permeability pattern (Wallow and Engerman, 1977; Leuenberger and Babel, 1978). Wallow and Engerman (1977) reported an increased permeability of certain retinal vessels to HRP in longstanding, intentionally poorly controlled, alloxan-diabetic dogs. The) suggested that this increased permeability was due to a junctional insufficiency. However, these vascular damages seem to be considerably more severe than the ones descriljed in this paper. In t,he present study, the vesicular transport in diabetic rats maintained for 2-6 months was greatly increased as compared with that in control rats and diabetic rats maintained for 1 month, Junctional transport of HRP between the capillary endothelial cells was occasionally found in diabetic rats maint,ained for 3-6 months. HRP seemed to reach the basement membrane through broken tight junct,ions. However, it was not clear which route-vesicular or junctional transport-predominantly contributed to the increased permeability. According to the freeze-fracture image of the strands in endothelial junctions of muscle capillaries, the strands were discontinuous in part (Simionescu, Simionescu and Palade, 1975; Wissig and Williams, 1978). It could be speculated that HRP passes between an interruption in the network of strands and transfers to the basement membrane. Reaction product was not limited to the basement membrane or to the extracellular spaces of the glial cells. This finding shows that the endothelial cells and the intercellular junction of endothelial cells play an important role in the barrier function, compared with the surrounding basement membrane and the glial tissue. In capillaries showing no thickening of the basement membrane an increased permeability was found, so this increase in permeability of the endothelial layer preceded the thickening of the basement membrane. Our experiment,s have not ruled out the possibility of a specific effect of streptozotocin. Pretreatment with 2-deoxyglucose or 3-o-methylglucose prior to streptozotocin injection (Ganda, Rossini and Like, 1976) seems to be a better control than just saline injection. But no increased permeability was found in the diabetic rats maintained for 1 month in the present study. Direct. toxicity of streptozotocin could not be the cause of an increased vascular permeability in the diabetic ra,ts maintained for 2-6 months. It is known that HRP induces an increased vascular permeability when injected locally or systemat#ically in rats (Cotran and Karnovskp, 1967 ; Cotran and Karnovsky, of 1968). According to our unpublished observations, intravenous administration 20 mg/lOO g of HRP did not seem to induce an increased vascular permeability in Wistar-King A strain ra,ts. Based on the present experimental results, it seems reasonable to postulate that disruption of the blood-retinal barrier in retinal capillaries of streptozotocin-diabetic rats is due to an increase in vesicular transport and a junctional insufficiency hehween
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the endothelial cells. The increase in capillary permeabilit’y precedes the thickening of the basement membrane and may play an important role in the development ot diabet,ic retinopathy. REFERENCES Babel, J. and Lcuenberger, P. (1974). A long term study on the ocular lesions in streptozotocitl diabetic rats. Albrecht v. Graefes Arch. klin. exp. Ophthal. 189, 191-209. Bloodworth, J. M. B. and Molitor, D. L. (1965). Ultrastructural aspects of human and canine diabetic retinopathy. Invest. Ophthalmol. 4, 103748. Cotran, R. S. and Karnovsky, M. J. (1967). Vascular leakage induced by horseradish perosidase in the rat. Proc. Sot. Exp. Biol. Med. 126, 557-61. Cotmn, R. S. and Karnovsky, M. J. (1968). Resistance of Wistar/Furth rats to the mast celldamaging effect of horseradish peroxidase. J. Histochem. Cytochem. 16, 382-3. Cunha-Vaz, J. G., Shakib, M. and Ashton, N. (1966). Studies on the permeability of the bloodretinal barrier. I. On the existence, development, and site of a blood-retinal barrier. Br. J. Ophthalmol. 50, 441-53. Cunha-Vaz, J., De Abreu, J. R. F., Campos, A. J. and Figo, G. M. (1975). Early breakdown of the blood-retinal barrier in diabetes. Br. J. Ophthdmol. 59, 649-56. Ganda, 0. P., Rossini, A. A. and Like, A. A. (1976). St.udies on streptozotocin diabetes. Diabetes. 25,595603. Graham, R. C. and Karnovsky, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302. Leuenberger, P., Cameron, D., Stauffacher, W., Renold, A. E. and Babel, J. (1971). Ocular lesions in rats rendered chronically diabet,ic with streptozotocin. Ophthnl. Res. 2, 189-204. Leuenberger, P. M. and Babel, J. (1978). Retinal microangiopathy in streptozotocin diabetic rats. Cellular and biochemical aspects in diabetic retinopat,hy. INSERM Syw~posium. 7, 41-56. Papachristodoulou, D. and Heath, H. (1977). Ultrastructural alterations during the development of retinopathy in sucrose-fed and streptozotocin-diabetic rats. Exp. Eye Res. 25,371-84. Rakieten, N., Rakieten, M. L. and Nadkarni. RI. (1963). Studies on the diabetogenic action of streptozotocin. Cancer Chewlother. Rep. 29, 91-8. Shiose, Y. (1970). Electron microscopic studies on blood-retinal and blood-aqueous barriers. Jap. J. Ophthalmol. 14, 73-87. Simionescu, M., Simionescu, N. and Palade, G. E. (1974). Morphometric data on the endot,helium of blood capillaries. J. Cell Biol. 60,128-52. Simionescu. M., Simionescu, N. and Palade. G. E. (1975). Segmental differentiations of cell junctions in the vascular endothelium. J. Cell BioZ. 67, 863-85. Sosula, L.. Beaumont, P., Hollows, F. C. and Jonson, K. M. (1972). Dilatation and endothelial proliferation of retinal capillaries in streptozotocin-diabetic rats: Quantitative elect,ron microscopy. Invest. Ophthalmol. 11, 926-35. Taniguchi, Y. and Nomura. T. (1968). Fine structure of retinal blood vessels in human diabetics. Acta Sot. Ophthalmol. Jap. 72, 1165-78. Wallow, I. H. L. and Engerman, R. L. (1977). Permeability and patency of retinal blood vessels in experimental diabetes. Invest. OphthaZmoZ. 16, 447-61. Waltman, S., Krupin, T., Hanish, S., Oestrich, C. and Becker, B. (1978). Alteration of the bloodretinal barrier in experimental diabetes mellitus. Arch. Ophthdmol. 96,878-g. Wissig, S. L. and Williams, M. C. (1978). Permeability of muscle capillaries to microperoxidase. J. Cell Biol. 76, 341-59.