Anatomy of the Mouse Retina. Capillary Basement Membrane Thickness

Transcription

1 Anatomy of the Mouse Retina. Capillary Basement Membrane Thickness R. A. Cuthberrson and T. E. Mandel Transmission electron microscopy of ultrathin sections was used to measure mouse retinal capillary basement membrane thickness (BMT). BMT increases predictably with age from 50 ± 9 nm at 6 weeks to 154 ± 27 nm at 20 months, but is not affected by strain or sex. There is an effect of retinal site, however, with BMT increasing with the radial distance from the optic nerve, 41 ± 4 nm (center), 54 ± 5 nm (mid-zone), and 64 ± 6 nm (periphery). The layer of retina from which capillaries were taken had no effect on BMT, and the inner and outer BMT maintained a consistent ratio, even at different ages. Invest Ophthalmol Vis Sci 27: , 1986 After the initial observation by Friedenwald 1 ' 2 that thickening of vascular basement membranes was a common feature of both diabetic retinopathy and nephropathy, capillary basement membrane thickness (BMT) has been used to quantify diabetic microangiopathy in a variety of tissues in several species. 3 " 10 Siperstein's 1 ' measurement of human muscle capillary BM started an extensive debate 12 " 14 over the methodology and interpretation of BM thickening, and led to the careful and comprehensive work of Williamson et al 14 in human skeletal muscle, and 0sterby, 15 Gundersen, 16 and Rasch 17 in the rat and human kidney. Efforts have been made to rationalize and standardize the stereological methods used to measure BMT by 0sterby et al 1516 in rat glomerular capillaries. The importance of establishing normal values for capillary BMT, both within particular species and between different tissues within the one species, has recently been emphasized by Steffes et al, 18 when they highlighted substantial differences between kidney and muscle capillary BMT in humans. Other studies in normal animals of BMT in the rat retinal capillaries especially those of Sosula 19 ' 20 placed the use of the rat retinal vasculature as a model of eary diabetic microangiopathic change on a firm foundation. The findings of Sosula 20 were important, as they stressed the influence of age, strain, sex, retinal area and retinal layer sampled in producing From The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia. Supported by grants from the National Health and Medical Research Council of Australia, Gavemer Foundation, and Perpetual Executors and Trustees Association of Australia. Submitted for publication: August 28, Reprint requests: R. Andrew Cuthbertson, MBBS, BMedSci, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia. variation in BMT in normal animals, which needed to be understood before any credible assessment of changes induced by, e.g., streptozotocin (STZ) diabetes could be made. Work by Fischer and Gartner 21 ' 22 in the rat retina distinguished the thickening of "inner" from "outer" BM, with the thickening in STZ-induced diabetes occurring disproportionately in the latter. The mouse offers a number of advantages to eye researchers. 23 The benefits of large numbers, inbred strains, rapid maturation, and rapid metabolic rates have produced a lot of recent work, especially in the field of diabetes. But the murine work has not been based on the same firm foundations as the rat model. 19 ' 20 In this study, we, therefore, defined values for murine retinal capillary BMT at five ages during the life span of the normal mouse, using two genetically dissimilar strains and both sexes. We also examined the influence of retinal area (center, mid-zone, and periphery) on mean BMT and its variance, as well as the influence of depth within the retina at which the vessel was found. Some of these variables did produce significant effects, which, if not controlled for, could lead to fallacious conclusions. Many workers have tried to develop a scientifically sound, practicable, and reproducible method of measuring BMT while accounting for the effect of tangential sectioning. 11 ' 12 ' 14 " 16 ' 21 ' 22 ' 24 Few of the theoretical models published deal with the physical realities of the highly magnified structure being measured in the electron microscope. The edges of tangetially cut BM are not absolutely electron-opaque and, thus, schematic attempts to model these structures as "cut pipe" may be misleading. 24 We describe a practical means of measurement, and experimental proof, which allows us to quote reliable mean BMT. 1653

2 1654 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1986 Vol. 27 Materials and Methods Basement Membrane Thickness Animals were maintained at The Walter and Eliza Hall Institute of Medical Research and were treated in accordance with the ARVO Resolution on the Use of Animals in Research. CBA mice were sacrificed at 6 weeks, and 4, 8, 12, and 20 months of age, (two s and two s at each age). Two BALB/c mice were also sacrificed at 6 weeks, 8 months, and 20 months. Animals were killed by cervical dislocation, and the right and left eyes were immediately enucleated and immersed in Karnovsky's fixative for 20 min to facilitate cutting. The eyes were then cut equatorially and the lens removed. Two wedges of retina, choroid, and sclera were cut with the apex of the wedge at the optic nerve head, and a base 2 mm wide. These wedges were further fixed for 1 hr, and then washed in cacodylate buffer overnight. The specimens were processed in 2% osmium tetroxide in cacodylate buffer for 2 hr, followed by aqueous uranyl acetate for 2 hr, before dehydration in serial acetones. The retinal wedges were, of course, originally curved. It was possible, however, to flat embed them in Spurr's resin, with minimal distortion, due to the narrowness of the wedge and the viscosity of the embedding medium. Blocks were coded on embedding and were thereafter treated 'blind'. The code was broken only after final measurements of BM thickness had been made. Sections were taken from the mid-zone of one of each pair of retinal wedges, and the depth within the retina at which a vessel was found was recorded. The second of the pair was only cut if sections were unsatisfactory from the first. In addition, the 6-week-old BALB/c mice also had sections taken from the center and periphery of the retina. Wedges from all the right eyes were cut, and, in CBA 8-month-old mice, both eyes were examined. One micrometer thick sections were taken from the appropriate area of the retina (center, mid-zone, or periphery) for orientation, and to ensure that at least ten capillaries were present. Ultrathin sections ( A) were then cut, stained with uranyl acetate and lead citrate, and examined in a Philips (Eindhoven, The Netherlands) EM300 transmission electron microscope. The microscope was calibrated before the experiment was begun. The first 10 capillaries, identified by morphology and size, were photographed at a fixed magnification, and the prints subsequently enlarged to give a final magnification of X29,000. Their depth within the thickness of the retina was also noted, being allocated to one of three retinal layer: A) nerve fiber layer (NFL) and ganglion cell layer (GCL); B) plexiform layer (IPL) and inner nuclear layer (INL), or C) outer plexiform layer (OPL). Each print was then placed beneath a plastic sheet on which 20 equally spaced radial lines had been drawn, and BM thickness was measured on a Zeiss (Oberkochen, West Germany) MOP 30 magnetic table where the radial lines intersected the BM. Both "inner" and "outer" BM were measured. These were defined, after Fischer and Gartner, 22 as "inner", the BM between the endothelial cell and pericyte; and "outer", the BM between either endothelial cell or pericyte and the surrounding glial tissue, respectively. Readings were only taken where the cell membranes bordering the BM were clearly visible as sharp black lines. Vessels were excluded from the study where less than 12 measurements could be made, and where the vessel was not a capillary on morphologic grounds. 25 A mean result for each vessel was then calculated, and, from these figures, a mean for each animal and each group. Thus the mean thickness expressed for any strain, sex, and age combination represents at least 240 measurements. Reproducibility of this method of measurement was tested by one observer assessing a series of masked prints at different times. Results were analysed where appropriate by linear correlation and 2-tailed Student's t-test, with P < 0.05 taken as significant. The measurement and analysis of BMT remains controversial," 1516 and our aim was to use a simple, well-described method of measurement, based on a testable theoretical model. Our technique for accounting for tangential sectioning centered on whether the trilaminar cell membrane was clearly visible on each side of the BM in question at X90,000 magnification. An ultrathin section of mouse retina was mounted in a Philips PW6500 goniostage, which enabled the specimen to be rotated and tilted about a selected axis. Using the goniostage at high magnification (X 90,000), we were able to focus the microscope on a tangentially sectioned segment of RCBM and then tilt the section until the trilaminar cell membranes bounding the BM were clearly seen. Thus we could assess the effect of degrees of tangential sectioning on both our ability to discern the trilaminar membrane and on the resulting apparent change in BMT. Other variables, such as outof-focus EM prints, were automatically excluded by this method. Clear trilaminar membranes at X90,000 appeared at a sharp black line at X29,000. Where the cell membrane detail was blurred at X90,000, the structure was ill-defined at X29,000. Results BM Thickness Figure 1 (top) shows the projected thickness of BM which happens to be sectioned tangentially to the Ion-

3 No. 11 BASEMENT MEMBRANE THICKNESS OF MOUSE RETINA / Curhberrson. and Mandel 1655 gitudinal axis of its vessel. Its thickness should, in theory, be greater than the "minimal BMT" of Williamson. 24 We took such a "blurred" vessel, Figure 1 (top), in retinal thin section mounted on a Philips goniostage and photographed the same length of basement membrane as the specimen was tilted. A tilt of at least 18 was required to sharpen the originally blurred cell membranes (Fig. 1, bottom), implying that the original section was cut tangentially at 18 from normal. More importantly, at these extremes of "tangential sectioning," the apparent BMT was not increased by more than 10%, and above ±18 from the point of sharpest focus, the measured BMT was, in fact, less than in the normal section. We, therefore, had a guide to the reliability of measurements taken at points where the trilaminar cell membrane is sharply seen at X90,000. At the lower magnification (X29,000) used for the routine BMT measurements, we found that the trilaminar membrane resolvable at X90,000 appeared as a sharp black line. Thus, we used the appearance of a single sharp cell membrane on each side of the BM as the criterion for measurement at X29,000. We analysed thefiguresobtained for BMT to determine whether mean BMT was significantly altered by age, strain, or sex. We further examined the effect of different areas of sampling in the retina (center/midzone/periphery), and of different depths of sampling (A, B, and C) on BMT. Finally, we asked whether the "inner" and "outer" BMs behaved in the same way, to the above changes. Controls of our quantification method came from assessing BMT in right eyes of duplicate animals and by comparing right and left eyes of the same animal. Fig. 1. (top) The mouse Section micknfiss retinal capillary basement membrane (X90,000) is Oblique section blurred due to tangential sec- of 22 tioning. This specimen was mounted on a Philips PW6500 gonioscopic stage. The schematic diagram shows the reason for the indistinctness of the trilaminar cell membranes at this degree of tangential sectioning. {bottom) The same area of Section basement membrane tilted thickness 22 to the point of maximal Tilt of 22 sharpness of the cell membranes, analogous to normal sectioning. Importantly, between these two extremes of cell membrane sharpness, the basement membrane thickness did not vary by more than ±10%, and at "tangential sections" more oblique than ±18, the BMT was, in fact, decreased. Table 1. Summary of retinal capillary basement membrane thickness in the mouse by age, sex, and strain* Age (months) CBA Strain BALB/c Strain Sex Mean BMT ±SDnm 47 ± 7 53±12 58 ±10 59 ±16 72 ± 9 77 ± ± ± ± ±26 55 ± 9 77 ± ±35 Mean BMT±SD nm, and pooled 50 ± 9 59±13 75 ± ±17 154±27 * Mean and SD expressed for two strains, each age group, and both sexes. All vessels measured for these data were taken from the mid-zone of the retina. There were duplicate animals within each subgroup. As can be seen from Table 1 and Figure 2, the BMT increased with age within subsets of strain and sex and as mean values pooled from all groups. There was a strong correlation between age and mean BMT (r > 0.95, n = 20, P < 0.01). The values increased from 50 ±9 nm at 6 weeks to 154 ± 27 nm at 20 months. Differences between the mean BMT of consecutive age groups were all significantly different. BMT was quite constant between strains of the same age and sex, when mean values of all three retinal areas were considered, and when the data was further analyzed to compare retinal layers. For example, when the three retinal layers were pooled, CBA and BALB/c s of 20 months of age had mean BMT of 152 ± 22 nm and 158 ± 30 nm, respectively. Similarly, comparing BMT in vessels from the same retinal area and the same layer within age-matched retinas, we found no significant difference between the sexes. Male 20-month-old CBAs had a mean mid-zone BMT of 158 ± 24 nm, compared with 152 ± 22 nm for matched s. When we compared BMT within a single strain, sex, and age combination ( 6-week-old BALB/c) between three areas of the retina (center, mid-zone, and periphery), we found a trend of increasing BMT as we moved radially out from the nerve head to the periphery (center 41 ± 4 nm, mid-zone 54 ± 5 nm, and periphery 64 ± 6 nm; center vs mid-zone P < 0.01, midzone vs periphery P < 0.02). These differences between the center, mid-zone, and periphery were significant, both when pooled as mean values and when further

4 1656 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1986 Vol. 27 within the various age, sex, and strain subsets. This ratio was further maintained when we compared center, mid-zone, and periphery of the BALB/c 6week-old retina, and when the three defined retinal depths were compared. The only exception to this observation was in the 20-month-old animals with exceptionally thick outer basement membranes, where this exaggerated thickening was not followed in proportion by the inner BM, giving a higher ratio, of 2.5 for CBA s and 2.0 for BALB/c s. The variation between mean BMT taken from the mid-zone of the right eye of the duplicate animals within each strain, sex, and age group were never significantly different (e.g., 6-week-old CBA: 45 ± 7 nm and 48 ± 7 nm; 20-month-old CBA: 148 ± 23 nm and 165 ± 36 nm). Finally, comparing right eye and left eye BMT from the mid-zone of single animal (BALB/c, 8 months old) gave results which were not significantly different (right eye 77 ± 9 nm, left eye 80 ± 8 nm). Discussion B Fig. 2. A, A typical retinal capillary (XI 4,000) from a 6-week-old BALB/c mouse. Pericyte nucleus (P) and endothelial cytoplasm (E) are shown. B, A retinal capillary (X 14,000) from a 20month-old animal of the same strain, showing "inner" (I) and "outer" <O) basement membranes, separated in this case by pericyte cytoplasm. At these extremes of age, the basement membrane thickening is obvious to the eye. divided into the three subsets by retinal layer (A, B, and C). Interestingly, there was also a trend for BMT to be greater in layer A than the deeper layers, although these differences just failed to reach statistical significance. This finding was consistent across sex, strain, and age divisions. For example, 6-week-old CBA s had mid-zone BMTs of 49 ± 6 nm in A, 45 ± 5 nm in B, and 41.0 ± 4 nm in C, with similar figures for the matched animals. The trend was seen in all age groups up to the 20-month-old BALB/cs, for example, where the values were A, 184 ± 28 nm; B, 151 ± 17 nm; and C, 143 ± 42 nm. The ratio of outer to inner BMT remains fairly constant at about 1.7 when examined at the mid-zone site, Much dispute, in the past, has been over the means of accounting for tangential sectioning of vessels and the consequent artifactual "thickening" of the BM in the longitudinal axis of the resulting ellipse. Many of the published methods have tried to reduce the coefficient of variation associated with these measurements and, thus, to increase the chance of detecting smaller statistically significant differences between groups of subjects Gonioscopic Stage Mathematical models of tangential sectioning usually emphasize the artifactual thickening associated with sections taken increasingly angled to the normal.15'24 We have shown, however, that within the range of ± 12 of the cell membranes being clear, these membranes remain in focus, and the BMT increases less than 10%. Above about ±12 of tilt, the cell membranes become less distinct, but, again, BMT is not significantly altered. Above ±18 (Fig. 1, top) the trilaminar membrane cannot be identified at X90,000, and, at X29,000, the cell membrane is indistinct, while the BMT is slightly decreased. Thus, applying the Siperstein "multiple measurement" 10 method, while only taking readings where the cell membranes were clearly in focus at X29,000, should give us a mean BMT for tangentially sectioned vessels within 10% of their true values. Basement Membrane Thickness Given the utility of using the BMT of the laboratory mouse as a means of quantifying retinal vascular pa-

5 No. 11 BASEMENT MEMBRANE THICKNESS OF MOUSE RETINA / Curhberrson and Mandel 1657 thology, 23 the importance of establishing normal values for these structures under the influence of strain, sex, age, and area and layer of the retina sampled becomes apparent. Steffes et al 18 have confirmed in the human that capillary BMT from different anatomic sites within the same species may not only be numerically different, but may behave quite differently in response to physiologic changes, such as aging, and pathologic states, such as diabetes. Comparing our findings in the normal mouse retina, with those of Sosula et al 20 in the normal rat retina, highlights the significant interspecies differences which exist. Murine retinal capillary BMT increases with age, independently of all other parameters studied, which is in agreement with the work of Bloodworth and Engerman 26 and Sima et al 10 in the rat. This striking increase over the relatively short lifespan of the animal suggests that the often intuitively held view that "aging" is accelerated in animals with a rapid metabolic rate is at least true in this instance. There were no effect of strain difference confirming the findings of Sosula et al 19 ' 20 in the rat. Similary, sex difference alone appeared to make no impact on BMT. We found that, within a single age, strain, and sex combination, the BMT increased centrifugally from the center. Thus, in studying pathological changes in BMT in the mouse, it is important to control for the site of sampling, as well as age of the animals under comparison. Analysis of the retinal layer from which the capillaries were sampled did not show a statistically significant effect on BMT, either overall, or within any specific age, strain, and sex subset. We did, however, find a trend of decreasing BMT with depth within the retina, which confirms Sosula's 20 finding that the mean thickness of outer BM of capillaries in the nerve fiber layer was significantly thicker than that of the inner plexiform layer and outer plexiform layer, with no significant difference between the last two layers. More recently, Sima et al noted a correspondingly increased thickening of RCBM from "superficial" compared with "deep" capillaries in the spontaneously diabetic BBrat, 10 although these layers were not defined. Fischer and Gartner 22 observed a disproportionate increase in the outer BMT in STZ-induced diabetes in the rat. We measured inner versus outer BMT where these structures lay opposite to one another. The ratio of outer to inner BMT was conserved across the other variables, indicating that, in the normal animal, the inner BM (although consistently thinner) increases in size with age in proportion to its matching outer counterpart; The only exceptions were the 20-month-old animals, where the grossly thickened outer BMs had increased out of proportion. We suggest that this parallel thickening of inner and outer BM should be taken into account when comparing these structures in, for example, experimental diabetes. Our confidence in the finding of increasing BMT with age in the mouse is supported by the tight correlations between right eyes of duplicate animals and between the right and left eyes of the same animals. However, even the mean of measurements from a single vessel has quite a high standard deviation, indicating the broad biological variability in the structure we were measuring, by the technique we had chosen. This standard deviation must affect any study which attempts to draw significance from experimental pathology. It also highlights the importance of stating the measurement and analytical techniques used to produce the data, as there is no doubt that the Williamson 24 method of measuring "minimal BMT" will reduce this standard deviation over the more random measurements made by the Siperstein" method. Published data 23 quoting a standard deviation using the Siperstein method, for mean BMT at 8 months of age in the mouse of ±5.5 nm, for such a highly variable biological structure need to be considered critically. BMT in the mouse may provide a means of accurately quantifying retinal microvascular pathology. Tangential sectioning is not as serious a problem as it might intuitively appear, if simple guidelines are followed. However, as we have shown in this study, the intrinsic variability in BMT within the one vessel and between vessels from the same retina provides a significant background which must be accounted for if statistically significant pathologic changes are to be stated. Future studies using the mouse BMT to quantify diabetic microangiopathy need to state the methods used to arrive at mean BMT values and to control for age, retinal area, and retinal layer of capillary sampling. Key words: retina, mouse, anatomy, capillary basement membrane thickness References 1. Friedenwald JS: A new approach to some problems of retinal vascular disease. Am J Ophthalmol 32:487, Friedenwald JS: Diabetic retinopathy. Am J Ophthamol 33:1187, Ashton N: Vascular basement membrane changes in diabetic retinopathy. Montgomery Lecture, Br J Ophthalmol 58: 344, Babel J and Leuenberger P: A long term study on the ocular lesions in streptozotocin diabetic rats. Albrecht von Graefes Arch Exp Ophthalmol 189:191, Bloodworth JM, Engerman RL, and Powers KL: Experimental diabetic microangiography. I. Basement membrane statistics in the dog. Diabetes 18:455, Hori S and Mukai N: Ultrastructural lesions of pericapillary Miiller cells in streptozotocin-induced diabetic rats. AJbrecht von Graefes Arch Klin Exp Ophthalmol 213:1, ' Johnson PC, Brendel K, and Meezan E: Human diabetic peri-

6 1658 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1986 Vol. 27 neural cell basement membrane thickening. Lab Invest 44:265, Leuenberger PM, Beauchemin ML, and Babel J: Experimental diabetic retinopathy. Arch Ophthalmol (Paris) 34:289, Papachristodoulou D and Heath H: Ultrastructural alterations during development of retinopathy in sucrose-fed and streptozotocin-diabetic rats. Exp Eye Res 25:371, Sima AAF, Chakrabarti S, Garcia-Salinas R, and Basu PK: The BB-rat an authentic model of human diabetic retinopathy. Curr Eye Res 4:1087, Siperstein MD, Unger RH, and Madison LL: Studies of muscle capillary basement membranes in normal subjects, diabetic and pre-diabetic patients. J Clin Invest 47:1973, Kilo C, Vogler N, and Williamson JR: Muscle capillary basement membrane changes related to aging and to diabetes mellitus. Diabetes 21:881, Vracko R, Pecaroro RE, and Carter WB: Overview article: Basal lamina of epidermis, muscle fibres, muscle capillaries, and renal tubules: changes with aging and in diabetes mellitus. Ultrastruct Pathol 1:559, Williamson JR and Kilo C: Current status of capillary basement membrane disease in diabetes mellitus. Diabetes 26:65, sterby R: Quantitative electron microscopy of the glomerular basement membrane. A methodological study. Lab Invest 25: 15, Jensen EB, Gundersen HJG, and 0sterby R: Determination of membrane thickness distribution from orthogonal intercepts. J Microsc 115:19, Rasch R: Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Glomerular basement membrane thickenss. Diabetologia 16:319, Steffes MW, Sutherland DER, Goetz FC, Rich SS, and Mauer SM: Studies in kidney and muscle biopsy specimens from identical twins discordant for type I diabetes mellitus. N Engl J Med 312:1282, Sosula L, Beaumont P, Hollows FC, and Jonson KM: Dilatation and endothelial proliferation of retinal capillaries in streptozotocin-diabetic rats: Quantitative electron microscopy. Invest Ophthalmol 11:926, Sosula L, Beaumont P, Jonson KM, and Hollows FC: Quantitative ultrastructure of capillaries in the rat retina. Invest Ophthalmol 11:916, Fischer F and Gartner J: Morphometric analysis of basal laminae in rats with long-term streptozotocin diabetes. I. Vitreoretinal juncture. Exp Eye Res 34:595, Fischer F and Gartner J: Morphometric. analysis of basal laminae in rats with long-term streptozotocin diabetes. II. Retinal capillaries. Exp Eye Res 37:55, 1983.' 23. Rodrigues M, Currier C, and Yoon J: Electron microscopy of neural and ocular changes in virus-induced diabetes mellitus in mice. Diabetologia 24:293, Williamson JR, Vogler NJ, and Kilo C: Estimation of vascular basement membrane thickness. Theoretical and practical considerations. Diabetes 18:567, Hogan MJ and Feeney L: The ultrastructure of the retinal vessels. II. The small vessels. J Ultrastruct Res 9:29, Bloodworth JM, Engerman RL, Camerini-Davalos RA, and Powers KL: Variations in capillary basement membrane width produced by aging and diabetes mellitus. Adv Metab Dis (Suppl) 1:279, 1970.