prenatal and postnatal growth of the human descemet's membrane

Prenatal and Postnatal Growth of theHuman Descemet's Membrane

Collin Murphy, Jorge Alvarado, and Richard Juster

The origin, growth in thickness, and differentiation of Descemet's membrane was studied by light,electron microscopic, morphometric, and statistical methods in 67 specimens from 12 weeks ofgestation to 98 years. Descemet's membrane is formed by three major processes: growth in thicknessduring the prenatal period, prenatal differentiation into a striated basement membrane, and growth inthickness during the postnatal period. The initial step is the synthesis of an ordinary basementmembrane, which is very thin and quite different in appearance from the adult Descemet's membrane.Growth of the prenatal Descemet's membrane then proceeds by deposition of a series of similar"membrane units," which are stacked to form a lamellar structure consisting of at least 30 layers bythe end of gestation. Second, during prenatal life, differentiation of the membrane leads to theformation of a striated structure through the gradual addition of short and thin cross-linking bridgesseparated by 110-nm intervals that are disposed in a plane perpendicular to the lamellae. The thirdprocess occurs in postnatal life when the membrane continues to grow in thickness by deposition ofa nonstriated, nonlamellar material posterior to the striated prenatal layer. Regression analysissuggests that prenatal growth proceeds at a rapid but variable rate best described by a "sigmoid-like" function of age. Postnatal growth, in contrast, proceeds in a predominantly exponential mannerbut at a slower pace than in the prenatal period. The low variability and large size of our set ofmeasurements make these data especially useful for comparisons with pathologic specimens. InvestOphthalmol Vis Sci 25:1402-1415, 1984

The possibility of using Descemet's membrane asan historical record of cellular activities and pathologicevents has been considered in recent studies.1"612 Inthese studies Descemet's membrane has been used ina manner analogous to that in which tree rings areanalyzed to assess climatic changes, as first noted byWaring.12 Thus, a disturbance in the appearance ofDescemet's membrane may suggest that a pathologicprocess has occurred. Localization of the abnormality,either in the anterior striated layer produced duringthe prenatal period or in the posterior nonstriatedlayer secreted during the postnatal period, can helpus to learn when the pathologic process begins. Forexample, developmental disorders affecting the cornealendothelium, such as in Peters' anomaly or in con-genital hereditary endothelial dystrophy, produce adisruption of the anterior striated layer.12 In otherhereditary disorders, such as in macular corneal dys-

From the Department of Ophthalmology, University of CaliforniaMedical Center, San Francisco, California.

Supported by NEI Grants EY 02068, EY 02162, EY 03903, byResearch to Prevent Blindness, Inc., and by That Man May See,Inc.

Submitted for publication: May 14, 1984.Reprint requests: Jorge Alvarado, MD, Department of Ophthal-

mology, U-490, University of California School of Medicine, SanFrancisco, CA 94143

trophy, inspection of Descemet's membrane revealsthat the cellular dysfunction actually commencessoon after birth, when an abnormal heparan proteo-glycan is secreted by the endothelial cells into theentire posterior, nonstriated layer.3 Here the datingtechnique is very useful in that we have learned thatthe basic disease process antecedes the clinical onsetof this disorder by several decades. In cases of Fuchs'endothelial dystrophy, membrane abnormalities ap-pear to arise at a variable time after birth,4 but inacquired disorders, such as in aphakic bullous kera-topathy, the abnormal membrane is secreted muchlater in life, most likely following cataract removal.5

An important limitation of this dating method isthat the date of onset of most diseases can be estimatedonly in fairly tentative terms. This lack of precisionmay be due in part to the great variability observedin the thickness measurements for normal mem-branes.6 This variability imposes constraints on theprecision of our dating estimates. Furthermore, onlytwo studies have measured the thickness of Descemet'smembrane as a function of age and these data setsappear to be quite different from each other. Frankl7

carried out the first morphometric study in 1969 with30 surgical specimens, and in 1983 Johnson et al6

studied 23 normal specimens obtained at autopsyand examined by light and electron microscopymethods. Because of inconsistencies between the two

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No. 12 GROWTH OF HUMAN DESCEMET'S MEMBRANE / Murphy er ol. 1403

Table 1. Specimen list

Age Sex

Fetal specimens (weeks)12161718181920222424262628

Postnatal specimens (years)

00000.230.500.921.001.081.50267

111516182121

F

M

MMFMMMF

MF

F

Thickness of Descemet'smembrane (p.m)

0.080.360.620.470.390.831.020.661.101.731.271.921.77

2.692.593.183.163.603.304.703.906.106.204.508.207.005.407.609.407.399.007.30

Sp. no.

143555OC

2020208520142018410286C554C551C

2003558C559C

287C568C282C283C572C617C565C444543C395476C574208475C409C311C469C312C289C

Age Sex

Postnatal specimens (years)2224252629293031323337414344485155555859606063636468696978788183889398

FM—FMMFFMFFMFM

FMMMFFFFFMFMFFMF

MMF

Thickness of Descemet'smembrane (nm)

9.007.80

10.209.406.40

11.009.40

11.409.609.00

11.209.60

11.409.607.40

10.2010.808.608.80

12.609.00

13.9013.8011.0012.6015.0014.0012.6016.0011.4019.8017.6018.0013.9017.60

Sp. no.

416C409338C383602C391C315C194C36074560041IC257C447573C431709126C389C387564

2223274708948410C127C585427C267C260C593C601C405C538C

data sets, it is not possible to establish a single growthfunction and the lack of such a normal standardeffectively prevents making the proper comparisonswith tissues from pathologic specimens.

The purpose of our investigation is to provide acomprehensive standard derived from measurementsof 67 specimens from 12 weeks of gestation to 98years of age. We also have used light and electronmicroscopy to study the three processes involved inthe formation, growth and differentiation of Desce-met's membrane. Statistical analysis of our datashows there is little variability in the thickness mea-surements and that sufficient data has been gatheredto estimate the growth rate of Descemet's membrane.These data may permit a more precise determinationof the time of onset of events that lead to the secretionof abnormal Descemet's membrane by the cornealendothelium in various disease processes in the pre-or postnatal periods.

Materials and Methods

We collected 67 ostensibly normal corneas (Table1). Thirty-nine specimens (23 surgical specimens,

[where the eye had been removed due to the presenceof an orbital tumor, in which case the eye itself wascompletely normal, or a posterior melanoma, inwhich ophthalmic examination revealed a normalcornea and anterior segments] three specimens withnormal corneas from the renal bank of our institution,and 13 fetal specimens) were fixed promptly. Theother 28 specimens (all normal corneas suitable fortransplantation obtained from the regional eye bank)were fixed 1 hr to 3 hr after death. Standard methodspreviously reported were used for fixation and tissueprocessing of the central cornea.89 Informed consentwas obtained prior to the study from patients orrelatives according to usual procedures.

Sectioning of the araldite-embedded tissue was ina meridional plane perpendicular to the corneal sur-face. The perpendicular alignment was empiricallyverified as described previously.8 One-micron sectionsof Descemet's membrane and adjacent corneal tissuewere photographed at X250 with a calibrated Zeissphotomicroscope. Overlapping light micrographs wereenlarged to XI667 and a montage of each specimenwas assembled. These photographs allowed for an


1404 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / December 1984 Vol. 25

- % •

• • • - ^

Fig. 1. Stages in development of Descemet's membrane. Stromal tissue is at upper portion of each micrograph, endothelium below.Descemet's membrane is between arrow heads. Electron micrographs, X2O,75O. A, A 12-week fetus. Note that a thin but continuousmembrane is present. It consists of an electron-lucent layer adjacent to the endothelium and an electron-dense layer. B, A 16-week fetus.Approximately three electron-dense layers are present, separated by electron-lucent layers. C, A 19-week fetus. The membrane consists ofapproximately five lamellae. Note cross-linking bridges {single arrows) associated with narrow interlamellsir zones; wider interlamellar zonedevoid of cross-linking bridges (double arrows). D, A 26-week fetus. Here the membrane consists of about 10 lamellae; many cross-linkingbridges are evident (single arrows).

easy identification of the borders of Descemet's mem-brane. We computed the average of three measure-ments of thickness made at 20-cm intervals on themontage. In the prenatal specimens, measurementswere made on electron micrographs of thin sectionstaken at X83OO and printed at X20,750. On eachmicrograph, 20 measurements at 1 -cm intervals weremade and the mean recorded. All measurements ofthe thickness of Descemet's membrane on light andelectron micrographs were made from the anteriorboundary of Descemet's membrane adjacent to thestroma to its posterior boundary adjacent to theendothelial cell layer. In addition to these measure-ments, 23 postnatal specimens representing each de-cade of life were studied by transmission electron

microscopy (TEM). Here we recorded the averagethickness of the anterior striated layer from its anteriorboundary at the stroma to the posterior margin wherethe nonstriated postnatal layer begins, computed fromthree measurements made at 8-cm intervals on eachelectron micrograph. For TEM we used a JEOL 100Celectron microscope calibrated on a daily basis witha carbon calibration grid (54,864 lines/inch).

We studied the relationship between the thicknessof these tissues and age by using regression methods.Both linear and nonlinear models were examined,and we describe below a nonlinear model that seemsto describe the data for Descemet's membrane best.In addition, we also analyzed the data previouslypublished by Frankl et al7 and Johnson et al6 and


No. 12 GROWTH OF HUMAN DESCEMET'5 MEMDftANE / Murphy er ol. 1405

Fig. 1. E, A 26-week fetus. This specimen has several more layers of membrane and narrower electron-lucent zones between lamellaethan the specimen illustrated in D. F, Term infant. The entire membrane consists of compacted lamellae and is striated throughout.

compared their results with ours using regressiontechniques.

To gain some information as to how fixation andtissue processing alter the dimensions of a livingtissue, we examined the corneal stroma, in whichdimensions in vivo are well known. Our thicknessmeasurements of the corneal stroma based on histo-logic methods are compared with those reported fromin vivo measurements10 to see if tissue swelling orshrinkage may have occurred due to tissue processing.Similarly, we also compared measurements made ofthe corneal epithelial thickness with those reportedfrom in vivo measurements.'' The thickness of Bow-man's layer also was measured. Multiple regressionmethods, which allow us to account for the effect ofage on thickness, and t-tests or Scheffe's multiplerange test, as appropriate, were used to determinewhether the differences in thickness were related to

the rapidity of fixation, to the specimen source (ie,surgical, eyebank, or renal bank), or to sex.

ResultsOur studies of Descemet's membrane are reported

by describing first the three major processes involvedin the formation of this unique basement membrane.The first two occur in prenatal life, and the third isa postnatal process. Two processes are related specif-ically to the growth in thickness of this membrane,and one process involves its differentiation into astriated structure. Lastly, we provide estimates of thegrowth function of the membrane during the prenataland postnatal periods.

Formation of Descement's Membrane

A continuous membrane can be observed in thespecimen shown in Figure 1A, which was obtained


1406 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / December 1984 Vol. 25

by the twelfth week of gestation. Such early mem-branes are composed of an electron-lucent zone (37.3nm thick) adjacent to the endothelial cell membraneand an electron-dense band (36.7 nm thick) near thecorneal stroma. By way of comparison, the fetalcorneal epithelial basement membrane is composedof two layers with similar thickness and appearance(lamina lucida 39.7 nm; lamina densa 41.7 nm).8

Thus, Descemet's membrane starts out as an ordinarybasement membrane, which looks quite differentfrom the adult membrane. The further growth of thismembrane proceeds by a most unusual processwhereby a series of individual "membrane-units" aresecreted in a sequential manner, to form an extraor-dinarily thick and multilayered structure (Figs. 1B-F). This process proceeds rapidly so that while onlyone layer is present by 12 weeks (Fig. 1A), about tenseparate layers can be counted by the end of thesecond trimester of gestation (Fig. ID), and 30-40layers may be present by birth (Fig. IF).

At the same time that Descemet's membrane isbecoming thicker, it is also undergoing differentiationinto a striated basement membrane. This changeinvolves the appearance of short (approximately 170nm) and thin (approximately 40 nm) linear filamentsdisposed perpendicularly between the electron-denselayers (Figs. 1C-G). These structures can be seen asearly as the 16 week stage; however, it is easiest toobserve them midway during the second trimester,as shown in Figure 1C. Regions of the membranethat have only a few or no filaments have a relativelywide (approximately 40 nm) electron-lucent layer,while regions having numerous filaments tend to beconsiderably narrower (approximately 20 nm) (Figs.1C, D). This suggests to us that the filaments areactually cross-linking bridges, which physically bindand pull together adjacent electron-dense bands. Laterin development, the bridges are uniformly distributedand separated by a distance of 110-120 nm, whichimparts a characteristic banding pattern to the prenatalDescemet's membrane. During the last trimester ofgestation (Fig. IE), there is even further compactionof the striated anteriormost layer, which may berelated to a concomitant shortening of the cross-linking bridges or filaments. Although the membranestill maintains a laminated appearance it becomes

increasingly more difficult to discern the electron-lucent spaces separating the layers late in gestationor at birth (Figs. IF, 2A) in the four specimens westudied.

After birth (Figs. 2B-E), Descemet's membranecontinues to thicken by a third process which resultsin the deposition of a nonstriated, nonlamellar ho-mogeneous material. This material forms a secondlayer within Descemet's membrane referred to as thepostnatal layer, which can be clearly distinguishedfrom the striated prenatal layer in postnatal specimensof all ages.

The postnatal layer is usually rather homogeneousand uniform in appearance; however, we also observedsome irregularities in a few specimens. These consistof scattered fibrillar collagen (30 nm in diameter byat least 600 nm in length) (Fig. 2F) or a few areasfilled with a "wide-spacing" collagen (Fig. 2G). Thelatter inclusions are formed by aggregates of short,thin rods with a periodicity of 110 nm, which havethe same dimensions as the cross-linking bridges ofthe anterior prenatal layer. However, unlike thebridges of the prenatal layer, the orientation of thesesegments appears to be at random with respect to theplane of sectioning. Occurrence of this material inisolated lacunae within the postnatal layer suggeststhat the corneal endothelial cells have occasionallyreverted to synthesis of the prenatal crossbridges.Some of the inclusions we describe also exist in the"posterior collagenous layer",12 in Hassal-Henlewarts,1314 cornea guttata and Fuchs' endothelial dys-trophy.15 However, in our specimens these materialsoccupy only a very small portion of the postnatallayer and are found only in specimens from elderlyand otherwise normal individuals.

Growth Rate during the Prenataland Postnatal Periods

The membrane thickness measurements of the 67specimens examined are shown in Table 1. Usingthese thickness measurements, we have employedregression methods to estimate the growth functionof Descemet's membrane (Table 2). These data areshown plotted by age in Figures 3A and B, alongwith our estimated function. Figure 3A contains datafrom conception to two years of age and Figure 3B

Fig. 2. Postnatal growth of Descemet's membrane. In micrographs A-E, the stroma is in the upper portion, Descemet's membrane isbetween arrowheads, and the endothelium is below. Electron micrographs. A, Term infant. Entire membrane contains striations. B, Age 11.The postnatal portion of the membrane is the unstriated homogeneous layer adjacent to the endothelium. This layer becomes thicker withage. Also note the thin, relatively unstriated portion of prenatal layer adjacent to stroma found in specimens over age 2 (arrows) (X 16,500).C, Age 29 (XI 6,500). D, Age 63 (XI6,500). E, Age 83. Note fibrillar inclusions within the very thick postnatal portion of membrane (arrows)(XI 6,500). F, Fibrillar inclusions in postnatal portion of membrane. Age 83. Note striations (X32,5OO). G, "Wide-spacing" collagenousinclusion in postnatal portion of the membrane. Age 63. Electron-lucent regions are oblique sections of fibrillar collagenous inclusions.(X32.5OO).



T

y


1408 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1984 Vol. 25

Table 2. Thickness of Descemet's membrane as afunction of age (N = 67 specimens, ages12 weeks gestation to 98 years)

lo&.(thickness) = 1.816 - 0.4889/(age)'5 + 0.011 ageSE = 0.045 0.0148 0.001

/>(t-test) < 0.0001 0.0001 0.0001F = 1171.15; / ' ( F test) < 0.0001; SE of the estimate = 0.19;

R2 = 0.97.

SE = standard error.

contains the data from subsequent years. The esti-mated function for the entire time period studied isshown in Figure 3C, along with estimated 95% con-fidence limits.

The estimated equation that best describes therelationship of thickness with age measured in yearsfrom conception, involves two components (Figure3). This equation can be expressed either as:

loge(thickness) = 1.816 - 0.4889/(age)'5 + 0.011 age

or as:

Thickness = e[1816 "

The first term of this equation, —0.4889/age15, indi-cates that the logg of thickness changes with age15 inan inverse manner; in other words, thickness can bedescribed as a sigmoid-like function of age as shownin Figures 3A and C. Due to this term, the rate ofgrowth in thickness starts out slowly during the earlyfetal period, accelerates, and then slows down begin-ning at about 20-weeks gestation. The decelerationoccurs as l/(age)'5 increases, and thickness approachesthe value e1816 (approximately 6.15 fim) asymptoti-cally. If only this first term, 1/age15, were actingthroughout life the curve would appear as illustratedin Figure 4A.*

The second term in the equation for Descemet'smembrane, 0.011 times age, represents the lineardependence of the log of thickness on age (Fig. 3B);in other words, this term represents an exponentialdependence of thickness on age. If only this exponen-tial term were acting throughout life, the curve wouldappear as in Figure 4B. However, the combined effectof these two components leads to a continued increasein thickness with age, beyond the 6.15-/im thresholdlevel determined by the sigmoid-like term alone. Ascan be seen from the shape of the function in Figure

* In this context, it is interesting to note that we found that theprenatal growth of both Bowman's layer and the corneal stroma iswell described by a similar sigmoid-like function. In both of thesetissues, thickness increases at a rapid pace during gestation, reachinga limit of about 650 fim for the stroma and 12 /im for Bowman'slayer soon after birth and remains constant thereafter (Figs. 5A, B;Tables 3, 4).

3C, where the combined effects are represented (ie,the entire estimated equation), the first componenttends to dominate the rate of growth during the fetaland early postnatal periods, whereas the second com-ponent tends to dominate later in life. Our finding

5

4

§35

2

-0.76 -0.4

Conception

0

Birth

20.0

17.5

15.0

g 125

o

^ 10.0

7.5

i I I I I

10 20 30 40 50 60 70 80 90 100

Age(Years)

Age(Yaars)

Fig. 3. A, Impact of age on thickness of Descemet's membranein 24 specimens, ages 12 weeks gestation to 2 years. B, Impact ofage on thickness of Descemet's membrane in 43 specimens, ages6-98 years. C, Impact of age on thickness in all 67 specimens, ages12 weeks gestation to 98 years. Predicted thickness for the entireestimated equation is represented by this regression curve. Ninety-five percent confidence limits for this curve are shown as well.These intervals represent the range of the "conditional mean ofthickness" for a given age. Within this area, we would expect tofind the "true" function (and not specific values) relating thicknessto age. This area should not be used for predicting a "normal"range for individual values.



o 35

0 10 20 30 40 50 60 70 80 90 100t . .

Birth Age (Years)

2.0

B0t

Birth

40 50 60

Age(YBars)

70 80 90 100

Fig. 4. A, Impact of age on thickness if only the first term of theregression equation described growth throughout life. B, Impact ofage on thickness if only the second term of the equation describedgrowth throughout life. The growth curve for the function consistingof both terms is shown in Figure 3C.

that the growth curve for Descemet's membraneconsists of two components seems to be consistentwith our histologic observations that two differentprocesses appear to be involved in membrane thick-ening; ie, formation of the prenatal striated layer andof the postnatal homogeneous layer. It is also impor-tant to note that the curve we have derived forprenatal and early postnatal growth of Descemet'smembrane resembles the growth curves found forother developing human systems; eg, fetal bodyweight,16 fetal head circumference,17 the length of theposterior segment in the eye,18 and the diameter ofthe cornea itself.919-20

A 12-week-old fetal specimen is the youngest thatwe studied and at this stage there already was acontinuous membrane present. Wiille and Lerche21

were able to examine a specimen in the eighth weekof gestation and found some evidence that the cornealendothelium had begun to secrete a low densitymaterial subadjacent to the basal cell membrane,which they interpreted to represent "the earliest stage

1200

1000

800

600

400

200

0 10 20 30 40 50 60 70 80 90 100AGE (YEARS)

25 r

20

15

10

B° 0 10 20 30 40 50 60AGE (YEARS)

70 80 90 100

4.5

4.0

3.5

3.0

2.5

2.0

1.51-

10 20 30 40 50 60

AGE (YEARS)70 80 90

Fig. 5. A, Impact of age on thickness of corneal stroma in 49specimens, ages 17 weeks of gestation to 93 years. Postnatalthickness does not change with age. Note resemblance to curve in4A. B, Impact of age on thickness of Bowman's layer in 47specimens, ages 17 weeks of gestation to 93 years. Postnatalthickness does not change with age. This curve also resemblesFigure 4A. C, Thickness of the striated prenatal layer in 23postnatal specimens. No definite relationship of thickness and agewas found.


1410 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1984 Vol. 25

Table 3. Thickness of the stroma as a function ofage (N = 49 specimens, ages 17 weeksgestation to 98 years)

loge(thickness) = 6.454 - 0.1169/(age)'5

SE = 0.038 0.0245P(t-test)< 0.0001 0.0001

F = 22.68; P (F test) < 0.0001; SE of the estimate = 0.24;R2 = 0.33.


of Descemet's membrane." When we measured thislayer on their published electron micrograph, wefound that it was about 0.03 fim in thickness. Thisvalue would fall below the lowest value entered onour graph (Fig. 3A) near a gestational age when itwould seem reasonable to expect synthesis of Desce-met's membrane to commence. Examination of laterstages in our specimens showed that the membranemeasures 0.08 fim in thickness by 12 weeks ofgestation, and that it grows rapidly thereafter. By 16weeks the membrane has grown over fourfold inthickness, measuring 0.36 fim. A 24-fold increase inthickness occurs from the 12-week stage to the 26-week stage when it measures 1.92 fim. By the end ofgestation it measures around 3 fim. This representsan average growth rate of about 104 nm/week, begin-ning at around the eighth week when secretion com-mences, until birth. This would correspond to anastonishing growth rate of 5400 nm/year.

Our equation predicts the thickness of the mem-brane at birth to be about 3.00 fim. Descemet'smembrane continues to grow rapidly in the earlypostnatal period, doubling its thickness by age 2 whenit measures around 6.0 fim. After age 2, its growth ispredominantly exponential with our equation pre-dicting a thickness of around 16.5 ^m by 90 years ofage. The postnatal thickness observations fall tightlyaround the regression curve; this low variability isindicated by the low standard error of the estimate(0.19). Also, the very high R2 value for these data(0.97) indicates that most of the variability in thicknessis explained by the equation we describe.

In order to see if the thickness of the prenatal layer

Table 4. Thickness of Bowman's layer as a functionof age (N = 48 specimens, ages 17 weeksgestation to 93 years)

loge(thickness) = 2.521 - 0.2586/(age)'5

SE = 0.044 0.0259P(t-test)< 0.0001 0.0001

F = 100.03; P (F test) < 0.0001; SE of the estimate = 0.28;R2 = 0.69.


changes after birth, we measured its thickness in 23specimens representing every decade of life from birthto 83 years. We found that the prenatal layer averages3.13 fim at birth, although these measurements rangefrom 2.59-3.18 fim in the four specimens we studied.As shown in Figure 5C, measurements of the striatedprenatal layer made in our postnatal specimens arealso variable, having an average thickness of 3.08with a standard deviation of 0.66 fim. Regressionanalysis indicates that the prenatal layer may undergosome postnatal thickening with age. However, thevariability, of the data makes it difficult to state withcertainty whether there is a significant trend in anydirection.f Our position at this time is that furtherstudies are required to determine what happens tothe prenatal layer after birth. It is possible that all thevariability in this layer can be attributed to individualvariations in thickness at birth and not to postnatalmodifications. In the only other study of the thicknessof the prenatal layer, Johnson et al6 found no apparentchange with age and our regression analysis of theirdata supports this contention (P < 0.6702 for the nullhypothesis that there is no change in thickness withage, or that the slope is zero), although this data isalso quite variable (mean thickness = 3.08; standarddeviation = 0.62 fim). In addition to measuring thick-ness, we also examined the ultrastructure of thisstriated layer in the postnatal specimens and noticeda minor change near the anterior boundary of theprenatal layer in all postnatal specimens over the ageof two. This change appears to represent an alterationof a portion of the prenatal layer, which loses part orall of its striations in a thin (approximately 0.41 /im)band immediately adjacent to the stroma. This bandcan be distinguished easily from the rest of theprenatal layer as shown in Figures 2B-E.

Our analysis indicates that Descemet's membranethickens greatly during the postnatal period. A similarextraordinary thickening occurs in the basementmembrane which forms the lens capsule, as shownby Fisher and Pettet.22 These authors observed thatthe lens capsule was about 3.5 /im thick at birth andabout 12 fim by age 60, or a growth of approximately8.5 fim in 60 years. In Descemet's membrane, wefound a similar amount of growth in our data, wherethickness increases by about 9.5 fim, from 3.0 fim atbirth to 12.5 fim, in the 60-year period. We found inanother study that the much thinner unilamellarcorneal epithelial basement membrane grows inthickness in a linear manner and adds about 0.18/xm of membrane material over the first 60 years of

f Intercept = 2.76 nm (P < 0.0001); slope or change in thicknessper year = 0.0086 nm (P < 0.0857); standard error of the estimate= 0.39 Mm; R2 = 0.1341.



life, increasing from 0.09 to 0.27 /um in that time.8

Thus, all three of these ocular basement membranesappear to increase in thickness three- to fourfold over60 years of postnatal life. Fisher and Pettet22 alsothought that the growth of the lens capsule was morerapid during the first decade compared to later inlife, which we have now shown to be true for Des-cemet's membrane since it doubles in thickness duringthe first two years of life.

As shown by the statistics for the Descemet'smembrane regression given in Table 2, both prenataland postnatal increases in thickness are highly signif-icant and suggest that the change in thickness isrelated to actual growth of the membrane due tocontinued deposition of membrane material at arapid rate throughout a lifetime. We also consideredthe possibility that this increase in thickness mayhave been related to artifacts such as swelling due todelayed fixation. However, when we used t-tests tosee whether there was a difference in thickness betweenrapidly fixed specimens and specimens where fixationwas delayed, taking age into account, there was nodifference between these groups. In other words, wefound no evidence to show that swelling may haveaffected those aged specimens differentially so as toproduce the described age-related increases in thick-ness. Instead we found that if such swelling occurred,it affected the specimens in a random manner. Thesame result was obtained when we used a Scheffe'smultiple range test to study the effect of specimensource (whether surgical, eye bank, or renal bank) orof sex.

Discussion

The examination of a large number of pre- andpostnatal specimens by quantitative morphometricmethods as well as by electron microscopy and sta-tistical methods has given us new information aboutthe origin, development, differentiation, and growthrate of Descemet's membrane throughout a lifetime.Synthesis of this unique and unusual basement mem-brane, known as Descemet's membrane, actuallybegins with the formation of a simple and ordinarybasement membrane, like that of most epithelial andendothelial layers, around the eighth week of gestation.Then, it begins to grow in thickness by the additionof many membrane units to form a laminated mem-brane. Wiille and Lerche21 thought that each addi-tional layer had formed on the stromal side bysecretion from stromal fibrocytes (keratocytes). Thisnotion was challenged by Smelser and Ozanics,23 whofelt it "much more likely" that each membrane unitor lamina is added by the corneal endothelial cells.Our studies show that there is a polarization during

the formation and further differentiation of the newlysecreted Descemet's membrane. The least-differen-tiated membrane components, consisting of thin seg-ments of a "low-density material" first appear nearthe basal surface of the corneal endothelium. Grad-ually, these segments become fused so that they forma thin but continuous membrane, which later becomesmore electron-dense and finally acquires a striatedappearance. New layers of membrane are added orformed from the endothelial side. These findings areconsistent with the idea that the corneal endothelialcells, rather than the keratocytes, are mainly respon-sible for the synthesis of these component membraneunits. In the developing chick embryo, Hay24"26 feltthat the corneal endothelial origin of Descemet'smembrane is supported by ultrastructural studies,which indicate that secretory organelles develop withinthe corneal endothelium at the time when Descemet'smembrane first appears in close proximity to theendothelial cell membrane. However, it is still possiblethat keratocytes participate in some manner in thesecretion of a collagenous or another material priorto the formation of the membrane units. For instance,Linsenmayer et al27 have proposed that in the chickembryo, type V collagen may be secreted very early,either by endothelial cells or by keratocytes. That thecorneal endothelium is by itself capable of secretinga Descemet's membrane is now well known, not onlyfrom studies of injury and wound healing28"31 butalso from in vitro investigations, where monolayersof corneal endothelial cells secrete a Descemet'smembrane. For example, cultured bovine cornealendothelial cells produce an extracellular matrix,which has been shown to contain collagens III, IV,V,32 fibronectin,33 and glycosaminoglycans.34

The deposition of multiple, single basement mem-brane units in a sequential manner leading to theformation of a laminated membrane is an unusualprocess that has been observed only for Descemet'smembrane and the lens capsule, two highly specializedand the thickest basement membranes known.35"37

The lens epithelium secretes its first membrane unitby the sixth week of gestation, and deposition ofadditional units proceeds rapidly throughout the pre-natal period.37 These membrane units are very similarin appearance and in thickness to those of the prenatalDescemet's membrane. Differentiation of the lami-nated, prenatal Descemet's membrane is mainly re-lated to the secretion of thin and short filamentsdisposed in a plane perpendicular to the long axis ofthe membrane. These filaments, or linking cross-bridges, provide the prenatal membrane with a striatedappearance, which is a unique characteristic of Des-cemet's membrane since these striations are not foundin the lens capsule or other basement membranes.



After birth, growth of Descemet's membrane ap-pears to proceed without the formation of singlemembrane units, lamellae, or deposition of the link-ing cross-bridges filaments. Instead, we find that anamorphous material is progressively deposited poste-rior to the striated prenatal layer. Therefore, thisportion of the membrane is nonstriated and nonla-mellar. Both the lens capsule and Descemet's mem-brane grow throughout postnatal life. In the lenscapsule, Seland38 has proposed that postnatal growthproceeds through the secretion of membrane units ina sequential manner, such as we have observed inthe prenatal period for Descemet's membrane. Ex-amination of previously published electron micro-graphs of the lens capsule35 show that in fact, aspostulated by Seland,38 the membrane has a laminatedappearance consistent with the notion that its growthis mediated by the addition of membrane units. Thismeans that different processes participate in the growthof the lens capsule and Descemet's membrane duringthe postnatal period and that there are significantdifferences in the manner in which Descemet's mem-brane and the lens capsule are synthesized in boththe pre- and postnatal periods of life.

The prenatal layer of Descemet's membrane mea-sures about 3.0 nm at birth, and this striated anteriorzone persists throughout life. The entire prenatallayer stains differentially with the usual histologicstains, and its distinctive striated appearance was firstrecognized in early electron microscopic studies.3940

Using celloidin sections, Frankl7 measured the thick-ness of the striated layer to be about 2.8 nm in aterm infant; using paraffin sections, Cogan and Ku-wabara28 estimated the thickness of the striated layerto be 10 nm at birth. Our present electron microscopicstudy clearly demonstrates that the thickness of thefetal layer averages only about 3 nm by birth, andthat the entire membrane at this stage consists ofstriated elements. While it has been thought that thisfetal layer persists relatively intact through the post-natal period,6'35'41 the variability we observed in ourdata prevents confirmation of that notion at thistime. We found one minor postnatal modification ofthe prenatal layer: after age 2, there is a thin (approx-imately 0.41 urn), electron-dense, homogeneous layerin the anteriormost portion of the membrane adjacentto the stroma. This layer may correspond to thenarrow (approximately 0.68 /zm) silver-staining band,described by Frankl7 in celloidin sections. Thus, theanterior striated layer appears to consist of two sub-zones later in postnatal life.

Each of the stages we have identified in the for-mation and differentiation of Descemet's membranemay be accompanied by the synthesis of a set ofcharacteristic proteins and other molecules. However,

we cannot yet identify the biochemical correlates ofthe anterior striated layer, its cross-linking bridges, orof the posterior homogenous layer, with regard to thepresence of specific collagenous and noncollagenousproteins as well as of proteoglycans. Most studies ofthe biochemical organization of Descemet's mem-brane have been conducted in other species.42"47 TypeIV collagen, which is a basement-membrane specifictype, as well as types II, V, and I, were reported instudies of the chick Descemet's membrane.42"45'25

Hendrix,45 in a ferritin-labeled antibody study, re-ported that the striations in Descemet's membraneconsisted of type II collagen. In one study of bovineDescemet's membrane, types IV and V collagen wereidentified46; however, Labermeier47 found only typeIV in preparations where the entire thickness ofDescemet's membrane had been separated cleanlyfrom associated tissues. Recently, desmosine andisodesmosine, normally cross-linking constituents ofelastin, were identified in a nonelastin component ofbovine Descemet's membrane.48 Sawada49 investigatedisolated bovine Descemet's membrane after selectivesolubilization in a combination study that involvedfreeze-etch replica methods, TEM, and biochemicalanalysis. He found that Descemet's membrane wascomposed of stacks of two-dimensionally arrangedlattices. These may correspond to the rows of lamellaein the anterior striated layer. These lattices appearedto be composed of four components that includedtwo types of noncollagenous constitutents, whichwere 80 nm round nodes and an amorphous material,as well as two collagenous components, which were120 X 25 nm rods and 5-10-nm diameter finefilaments. We are intrigued by the notion that these120 X 25 nm rods may correspond to the cross-linking bridges in the prenatal layer. To date, veryfew histochemical or biochemical studies have beenconducted with human material. Only one study hasaddressed the question of age-related biochemicalchanges in the human Descemet's membrane. Free-man et al50 found that there were changes in theproportions of certain amino acids with age, whichcould be related to the late changes in the ultrastruc-ture of Descemet's membrane reported here. Twoattempts to identify macromolecular species in thehuman Descemet's membrane have been reported.Newsome et al51 used fluorescent antibodies againstfibronectin and type IV collagen. With both antibod-ies, two positive regions occurred in anterior andposterior zones of the membrane, whereas a centralzone remained unstained. Lutjen-Drecoll et al52 founda similar staining pattern for type II collagen. Clearly,further studies are needed before correlation is possiblebetween the morphologic processes we have observedduring the growth and differentiation of Descemet's



membrane and synthesis of specific collagenous andnoncollagenous molecules.

The fact that the normal Descemet's membranecontinues to thicken in postnatal life was noted byHenle as early as 1866.53 Later investigators such asRother in 196654 and Daiker in 197055 presentedbrief descriptions of this process in light microscopicstudies. Cogan and Kuwabara in 197128 published aconvincing ultrastructural study that showed thick-ening of Descemet's membrane with age, comparinga 6-month fetus, and specimens from 11- and 70-year-old individuals, although they did not estimategrowth rate. Hogan et al in 197135 reported thatDescemet's membrane measured around 3-4 pm inthickness at birth and 10-12 nm by adult life. How-ever, Frankl in 19697 published the first quantitativestudy using morphometric methods. He measuredthe thickness of the membrane by light microscopyin 30 celloidin-embedded specimens with normalanterior segments that were obtained at surgery.These specimens were of ages ranging from birth to86 years, and the thickness data was plotted as afunction of age where the measurements for thefourth to sixth decades appeared widely scattered.These data suggested to Frankl that the postnatalincrease in thickness continued until age 55, whenhe felt that no further growth was apparent. Whenwe analyzed his plotted data points by regressionmethods, we actually found that contrary to hisimpression, his data is consistent with a model inwhich thickness increases subsequent to age 55. Al-though the observed thickness at birth from his data(approximately 2.8 /xm) is similar to our findings, weestimated from his data that by age 90, thicknesswould be only 7.9 fim, compared with the estimateof 16.5 nm based on our data. In 1982 a comprehen-sive, quantitative ultrastructural study of Descemet'smembrane was conducted by Johnson, Bourne, andCampbell.6 Here the thickness of the entire Descemet'smembrane as well as that of the anterior striatedlayer were measured in 22 normal specimens obtainedat autopsy, ranging in age from 8 months to 88 years.Using correlation analysis, they found a significantpositive association between age and thickness of theentire membrane, although they did not estimate agrowth function. According to our regression analysisof their data, we found that membrane thickness atage 90 is predicted to be 12.7 nm, which is greaterthan the 7.9 /xm that we found for the Frankl databut still lower than the 16.5 pm for our study. Wedo not know why the growth functions for thickeningappear to differ among the three data sets. Frankl'sdata consisted entirely of surgical specimens, whichpresumably were promptly fixed, and Johnson's, ofautopsy specimens that underwent delayed fixation.

Our data included both sources; fixation was carriedout comparatively rapidly in all of our specimens.Differences in measurement techniques—establish-ment of boundaries of Descemet's membrane, planeof sectioning, or microscope calibration—could alsoaccount for these disparities. Regardless of the differ-ences in thickness obtained, the basic finding ofthickening with age is supported by the data from allthree studies.

A limitation of these histologic studies is that thereare no in vivo measurements of Descemet's membranethat could be compared with the measurements ob-tained by histologic methods. However, such in vivomeasurements have been made for the entire thicknessof the cornea, which is predominantly composed ofstromal tissue, using pachometry.10 Thus, in order togain some information about possible relationshipsbetween measurements obtained by our methods andthese in vivo studies, we have also measured thethickness of the stroma, Bowman's layer, and thecorneal epithelium.56 The results for the stroma andBowman's layer are presented in Figures 5A and Band Tables 3 and 4. In contrast to Descemet's mem-brane, in these tissues we find that no substantivechange in thickness occurs after early postnatal life.The variability in the postnatal stromal data appearsto be related to swelling effects, since both the mean(643.46 Mm) and SD (169.36; n = 42) are much largerthan the values reported in the in vivo study wherecorneal thickness was 518 nm (SD = 32) for menand 529 pm (SD = 29) for women.10 For Bowman'slayer, the mean postnatal thickness is 12.65 /xm witha standard deviation of 2.94 nm (n =41), where thelarge SD suggests possible artifacts of tissue swelling.Moreover, we found that the postnatal data forthickness of Bowman's layer and of the stroma werefar more variable, with higher standard errors of theestimate and lower R2 values than we found forDescemet's membrane (Tables 3, 4). On the otherhand, the corneal epithelium has a relatively stablethickness after the age of 10, with a mean of 56.8Aim (SD = 8.10; n = 19), which is similar to the meanof 62.1 /xm (SD = 5.86) obtained from in vivostudies.'' This indicates to us that swelling may affectthe stroma and Bowman's layer more than is thecase for Descemet's membrane or the corneal epithe-lium. It is also worthwhile to note that we found noevidence for systematic swelling or shrinkage of Des-cemet's membrane, Bowman's layer or the stromaamong specimens in this collection obtained fromdifferent sources. Based on these observations, wefeel that our studies indicate that the thickening wehave observed in Descemet's membrane is not relatedto swelling; instead, we believe that the increase inthickness with age is primarily related to continued



synthesis of this membrane by the corneal endothe-lium.

The most important result of our study is that wehave been able to estimate for the first time thegrowth of the normal human Descemet's membraneas a function of age throughout life. Our applicationof regression methods to a series of observations hasenabled us to calculate both pre- and postnatal growthcurves, and to compare these curves with those wehave estimated for other basement membranes. Wehave found that rapid growth during the fetal periodcan be described by a sigmoid-like function, and thatpostnatal growth follows a predominantly exponentialpattern. Thus, we have quantiatively and accuratelycharacterized the normal growth of Descemet's mem-brane with age. This study has provided us with astandard for the growth of Descemet's membranewhich can be used for future comparison with patho-logic specimens.

Key words: human cornea, development and growth, thick-ness, morphometry, Descemet's membrane, stroma, Bow-man's layer, epithelium

Acknowledgments

The authors thank Michael Krasnobrod for photographicwork, Andersen Yun for technical assistance, and DianeKitchen and Sandra Davis for assistance in preparation ofthe manuscript.

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prenatal and postnatal growth of the human descemet's membrane

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