Structure and Composition of the Rodent Lamina Cribrosa
JOHN MORRISON a.b*,SUSAN FARRELL a,ELAINE JOHNSON', LISA DEPPMEIER =, C. G. M O O R E ~, AND E M I L I E G R O S S M A N N ~
a Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Department of Ophthalmology, Oregon Health Sciences University and b The Portland Veterans Administration Hospital and Medical
Center, Portland, OR, U.S.A.
(Received Columbia 6 May 1994 and accepted in revised form 2 August 1994)
To define the architecture and extracellular matrix composition of the lamina cribrosa in rodents, normal, adult pigmented rat and guinea pig eyes were frozen and sectioned for light microscopic immunohistochemistry. Antibodies specific for collagens I, HI, IV and VI, laminin, elastin, and chondroitin and dermatan sulfate proteoglycans were exposed to longitudinal and cross-sections of optic nerve heads and their binding distributions observed with the avidin-biotin-peroxidase complex technique. Cross-sections of the intraocular portion of the rat optic nerve head revealed a horizontally oval shape with distinct, vertically oriented, laminar beams. The guinea pig optic nervehead cross-section was circular, with randomly oriented beams. In both animals, collagens I, HI and VI were found throughout the laminar beams, along with elastin fibrils. Collagen IV and laminin antibodies deposited along laminar beam margins and within the beams, representing astrocytic and vascular endothelial cell basement membranes. Both animals showed evidence for dermatan and chondroitin sulfate-containing proteoglycans in all connective tissue structures of the nerve head. In the rat, chondroitin-4 sulfate proteoglycans appeared localized to the sclera and laminar beams. The rat and the guinea pig optic nerve head possess an identifiable lamina cribrosa with structural proteins nearly identical to that of the primate. Both animals may provide affordable alternative animal models for in vivo studies on the role of the lamina cribrosa in glaucomatous optic nerve damage.
The lamina cribrosa, a dominant structure in the optic nerve head, is thought to play an important role in glaucomatous optic nerve damage. Hypotheses for this role range widely from protecting optic nerve fibers (Radius, 1981 ; Quigley and Addicks, 1981 ; Quigley, Addicks and Green, 1981; Quigley, Hohman and Addicks, 1983) to being the instrument of final damage to the nerve fibers themselves (Quigley et al., 1980; Minckler and Spaeth, 1981; Maumenee, 1981). Regardless of the precise function of the lamina cribrosa, its composition is of great interest, since this will influence the mechanical behavior of the lamina and its ability to either protect or harm optic nerve fibers (Zeimer and Ogura, 1989).
Numerous studies indicate that primate laminar beams consist of several types of interstitial collagen with interspersed elastin fibrils (Hernandez, Igoe and Neufeld, 1986; Hernandez, Luo and Igoe, 1987; Morrison et al., 1988a, 1988b; Goldbaum, Jeng and Logemann, 1989). Collagen IV, laminin and fibro- nectin are found along blood vessels and the margins qof the laminar beams, representing endothelial cell and astrocyte basement membranes. Recent studies
Presented in part at the Association for Research in Vision and Ophthalmology Spring Meeting, Sarasota, Florida, 1993.
* For correspondence at: Casey Eye Institute, Oregon Health Sciences University, 3375 SW Terwilliger Blvd., Portland, OR 97201.
suggest that several types of proteoglycans are present throughout the laminar beams in distinct patterns, depending u p o n their content of chondroitin and dermatan sulfate and degree of sulfation (Caparas, Cintron and Hernandez, 1991; Sawaguchi et al., 1992a, 1992b; Morrison et al., 1994).
The biochemical composition and architecture of the lamina cribrosa has been reported to change during the glaucomatous process (Tengroth and Ammitzboll, 1984; Hemandez, Andrzejewska and Neufeld, 1990; Knepper et al., 1990; Hernandez, 1992; Sawaguchi et al., 1992b). However, it is uncertain on the basis of enucleated human specimens alone ff these alterations are primary factors in the glaucomatous process, or represent secondary changes. Analysis of eyes with experimental primate glaucoma indicate that elevated intraocular pressure itself may induce specific changes in the composition of the optic nerve head extracellular matrix (ECM) in otherwise normal eyes, including altered collagen and elastin fibrils and deposition of several macromolecules within the pores of the lamina cribrosa (Morrison, Dorman-Pease and Dunkleberger, 1990; Quigley, Dorman-Pease and Brown, 1991; Quigley, Brown and Dorman-Pease, 1991; Fukuchi et al., 1991).
Numerous further studies are required to under- stand fully the complexities of the ECM response to elevated intraocular pressure and to determine the relationship of the lamina cribrosa to nerve fibers in the glaucomatous process. This work will be expedited
FIa. 1. Rat tissue labeled with antibodies to collagen type I. Cross-section of intraocular optic nerve head (A) appears oval and is oriented horizontally, with vertically arranged laminar beams (arrow). Retrobulbar optic nerve (B) is round, larger and has randomly arranged septa. Horizontally oriented longitudinal section (C) cuts across several laminar beams (arrows), which in vertical section (D), are only seen occasionally (arrow). (Original magnification, 95 x ).
by the development of cost effective alternative animal models of pressure-induced optic nerve damage, allowing for extensive preliminary experimentation before final confirmation with expensive primate material.
Our interest in developing an inexpensive, readily available animal model of pressure-induced optic nerve damage has encouraged us to study the support structures of the rodent optic nerve head. Using immunohistochemistry, we have evaluated the archi- tecture of the rat and guinea pig lamina cribrosa and determined its composition, concentrating on several extracellular matrix macromolecules already described in the primate.
2. Materials and Methods
Eight adult 1-year-old Brown Norway rats and five pigmented 1-1.5-year-old guinea pigs were killed by exsanguination following intraperitoneal injection of a 1.5 mlkg -~ solution containing 5 ml ketamine (100 mg ml-X), 2.5 ml zylazine (20 mg ml-1), 1 ml acepromazine (10 mg ml-1), and 1.5 ml sterile water. The eyes, judged normal by direct, magnified in- spection, were immediately enucleated, lanced at the superior equator, soaked overnight in 10 % sucrose at 4°C and then immersed in OCT mounting media and frozen with 2-methyl butane chilled in liquid nitrogen. All animal experiments were performed in accordance
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FIG. 2. Guinea pig eye labeled with collagen I antibodies. Cross-section at the level of the sclera (A) shows circular nerve with laminar beams (arrows) emanating from an inferior fibrovascular stalk. A similar pattern appears posterior to the globe. Horizontal longitudinal section (B) demonstrates a multilayered lamina (arrows) that is less distinct than when viewed in cross-section and tapers anteriorly into the gllal columns (arrowhead). (Original magnification, 95 x ).
with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Cross-sections through the optic nerve head and retrolaminar optic nerve were cut 3 #m thick on a cryostat and studied for overall architecture and composition. In some eyes, either horizontal or vertical longitudinal sections were cut through the optic nerve head. All sections were collected on r-aminopropyl 1- ethoxysilane coated glass slides and stored at -80°C until use for immunohistochemistry.
Sections were first fixed in methanol for 15 min at 4°C and then washed in phosphate buffered saline
(PBS). Sections were incubated for 30 min in PBS with 1% bovine serum albumin (PBS/BSA) containing 20% normal serum from an appropriate species to block non-specific binding. Excess blocking serum was then removed and the sections were overlaid with primary antibodies diluted 1:100 to 1 : 5000 in PBS/BSA overnight at 4°C. Sections were then washed with PBS and overlaid for 30 min with biotinylated secondary antibodies (Vector Laboratories, Burlin- game, CA, U.S.A.) diluted 1:200 in PBS/BSA with 20% appropriate serum. Following a second wash, the slides were exposed to avidin biotin peroxidase complex (Vector Laboratories), mixed 1 : 140 for rabbit primary antibodies and 1:50 for mouse primary antibodies in PBS/BSA, for an additional 45 min and then developed in 0.05% 3,3-diaminobenzidine with 0"02% hydrogen peroxide in 20 mM Tris buffer, pH 7.2, for 3 rain. They were then counterstained lightly with hematoxylin, dehydrated and coverslipped for viewing with a Zeiss Axiofot light microscope.
Primary collagen antibodies consisted of goat IgG antibodies to collagen I, III and VI purchased from Southern Biotechnologies and rabbit IgG antibodies to collagen IV (BioDesign, Kennebunkport, ME, U.S.A,). Other antisera included rabbit IgG antibodies to purified laminin (Telios, San Diego, CA, U.S.A.) and, for rat tissue, goat IgG antibodies to rat alpha elastin and, for guinea pigs, rabbit IgG antibodies to human alpha elastin (both from Elastin Products, Owensville, MD, U.S.A.). Use of all of these antibodies has been documented and published previously in several organ systems, including the eye (Morrison et al., 1988a; Demarchez, Hartmann and Prunieras, 1987; Engvall et al., 1986; Wrenn and Mecham, 1987).
For chondroitin and dermatan sulfate proteoglycan studies, sections were incubated for 30 rain at 37°C in enzyme buffer (pH 7.2) consisting of 20 mM Tris, 50 mM SOdium acetate, 100 mM SOdium chloride, 0-01% bovine serum albumin (fraction V, Sigma, St. Louis, MO, U.S.A.), and 0.1 U m1-1 chondroitinase ABC (EC 4 .2 .2 .4 , from Proteus vulgaris, Sigma) or chondroitinase ACII (EC 4 .2 .2 .5 , from Arthrobacter aurescens, Sigma). All slides were then rinsed in PBS and processed for light microscopic immunohisto- chemistry using mouse monoclonal IgG antibodies to chondroitin-4 sulfate (4S) and mouse monoclonal IgM antibodies to chondroitin-6 sulfate (6S) (ICN Immuno- biologicals, Costa Mesa, CA, U.S.A.). These antibodies are well characterized and specifically recognize the carbohydrate stubs that remain attached to the proteoglycan core protein after enzymatic removal of dermatan and chondroitin sulfate side chains (Cater- son, Christner and Baker, 1985; Couchman, Cater- son and Christner, 1984; PoreUo and LaVail, 1986; Hageman and Johnson, 1987; Porrello, Yasumura and LaVail, 1987). Incubation with 4S antibodies following chondroitinase ABe digestion labels core proteins with both dermatan and chondroitin 4- sulfate glycosaminoglycans; while only chondroitin
130 J. M O R R I S O N ETAL.
TABLE I
Results of immunohistochemical staining of rat and guinea pig laminar beams and optic nerve septa with extracellular matrix protein antibodies
Rat Guinea pig
Laminar Laminar Antibody beams Septa beams Septa
Collagen + + + + I confluent confluent confluent confluent
Collagen + + + + III confluent confluent confluent confluent
Collagen + + + + VI confluent confluent filamentary* filamentary*
Collagen + + + + IV linear'l" lineart linear J- linear1"
* Filamentary labeling present throughout the structure. ~" Linear pattern suggested labeling of astrocyte and vascular endothelium basement membranes.
4-sulfate containing proteoglycans are labeled after ch6ndroitinase AC digestion.
Control experiments consisted of substituting cor- responding dilutions of goat IgG for goat antibodies to collagen I, III, VI and rat alpha elastin, rabbit IgG for rabbit antibodies to collagen IV, laminin and human alpha elastin, and mouse IgG1 for mouse antibodies to chondroitin sulfates.
3 . R e s u l t s
Optic Nerve Head Architecture
We found substantial architectural differences be- tween the rat and guinea pig with regard to the shape of the optic nerve head and the configuration of the lamina cribrosa. Within the sclera, the rat optic nerve head was horizontally oval, with the laminar beams oriented primarily vertically (Fig. 1). Posterior to the globe, the optic nerve was larger and circular, with randomly oriented septa. Vertical longitudinal sections revealed a narrow scleral canal with an apparently sparse lamina cribrosa. In contrast, horizontal longi- tudinal sections showed a wider scleral canal, and cut across numerous readily identifiable laminar beams, arranged in two to three layers.
The guinea pig optic nerve cross section appeared circular both at the levels of the sclera and posterior to the globe (Fig. 2). Laminar beams were randomly oriented, emanating from a central fibrovascular stalk. Viewed longitudinally, either vertically or horizontally,
the lamina cribrosa appeared multilayered with individual beams less prominent and distinct than when viewed in cross-section.
Composition of the Lamina Cribrosa (see Table I)
Control studies consisting of substituting appro- priate dilutions of non-immune immunoglobulins showed minimal label without any recognizable patterns in either species (Fig. 3).
Interstitial collagens I and III showed identical, confluent distribution across the laminar beams (Figs 1, 2 and 4). In the rat, label was confined to the laminar beams, while fine extensions were also seen tapering anteriorly into the glial columns in the guinea pig (Fig. 2). Collagen type VI appeared confluent in rat laminar beams, but was more filamentary in the guinea pig (Fig. 4).
Basement membrane components collagen type IV and laminin both demonstrated distinct linear distribu- tions along the margins of the laminar beams and within the beams, both as punctate depositions and in association with blood vessels (Fig. 5). Both species demonstrated immunopositive elastin fibrils in the laminar beams that were oriented parallel to the beams themselves (Fig. 6).
In both the rat and guinea pig, labeling for chondroitin-4 sulfate plus dermatan sulfate proteo- glycans revealed confluent labeling throughout the sclera and laminar beams as well as the retrobulbar optic nerve septa, pia mater and optic nerve sheath
RODENT LAMINA CRIBROSA :131
FIG. 3. Controls exposed to non-immune IgG in place of primary antibodies. Both rat (A) and guinea pig (B) demonstrate minimal label, with no specific pattern. (Orig- inal magnification, 9 5 × ).
(Figs 7 and 8). In the rat, labeling for chondroitin-4 sulfate alone resulted in staining of the lamina cribrosa and sclera, only. In contrast, while we saw chon- droitin-4 sulfate proteoglycan labeling in the guinea pig sclera, there was none in the lamina cribrosa.
Chondroitin-6 sulfate antibodies showed confluent labeling within the laminar beams of the rat optic nerve (Fig. 9). Guinea pig nerves labeled with these same antibodies produced a more linear pattern along the beam margins and blood vessels with scattered additional label within the beams.
4. Discussion
Previous studies on the architecture of the rat optic nerve head have yielded conflicting results. Early reports indicated variable quantities of lamina cribrosa
FIG. 4. Rat (A) and guinea pig (B) cross-sections labeled with collagen type III and collagen type VI respectively. Collagen III shows confluent label within laminar beams (arrows). In guinea pig laminar beams (arrows), collagen type VI appears more filamentary. (Original magnification, 370 x ).
at the level of the sclera (Nicholls and Tansley, 1938) (Tansley, 1956). More recently, Johansson, using standard histology and scanning electron microscopy of trypsin digested specimens, has stated that the lamina cribrosa of the rat consists of a sparse, single sheet of connective tissue (Johansson, 1987).
On the other hand, Hildebrand, Remahl and Waxman (1985) have described a more extensive lamina cribrosa in rats. They showed that the rat anterior optic nerve has a bottleneck configuration, divided into three regions: the neck, or optic nerve head, which is bordered by the choroid and sclera and devoid of myelinated axons; the conical transitional zone, with increasing myelination beginning at the posterior edge of the sclera; and the optic nerve proper, where the nerve is widest, due to myelination
132
FIG. 5. Antibodies to collagen type IV and laminin, shown in the rat (A) and guinea pig (B), respectively, produce distinct linear label of the laminar beam margins (arrows) and capillaries (arrowheads) within the beams. (Original magnifi- cation, 370 × ).
of all axons. Using transmission electron microscopy, they also described transverse vessels with wide, col- lagen-filled perivascular spaces and astrocytic borders. These structures, which probably correspond to the lamina cribrosa, were seen in the neck and transition zone of the nerve, but not in the optic nerve proper.
In general, our findings corroborate those of Hildebrand et al. (1985). However, we also noted a distinct orientation to the rat optic nerve head and laminar architecture. Viewed in cross-section, the rat optic nerve head has a unique, horizontally oval shape that persists to the level of the fully myelinated optic nerve proper. Interestingly, a similar shape has been noted in the squirrel optic nerve (Tansley, 1956), but not in previous studies on rats, which did not employ cross-section analysis.
J. M O R R I S O N ETAL.
F1fi. 6. Elastin fibrils (arrow) in guinea pig laminar beams appear oriented parallel to the long axis of the beam. (Original magnification, 925 × ).
The laminar beams of our specimens also demon- strated a consistent orientation. Not always associated with blood vessels, rat laminar beams were primarily oriented vertically, across the short axis of the nerve. As with the nerve head shape, this orientation did not extend into the nerve proper.
We also found the rat laminar beams to be more extensive and multilayered than those described by ]ohansson (1987). This discrepancy may reflect methodologic differences. The trypsin digestion and critical-point drying required for the scanning electron microscopic studies of ]ohansson (1987) may con- dense and collapse delicate laminar beams. In addition, Johansson's standard histologic preparations were performed on sagittal sections, corresponding to our vertically oriented longitudinal sections. As we have shown, this orientation has a low probability of intersecting laminar beams, which are primarily vertical, and would underestimate the total amount of laminar tissue. Other studies did not specify the orientation of sectioning (Nicholls et al., 1938; Tansley, 1956; Hildebrand et al., 1985). Thus, orientation is important when using longitudinal sections to evaluate the architecture of the rat optic nerve head.
With regard to the guinea pig, our results cor- roborate ]ohansson (]ohansson, 1987) and others (Furuta, Lindsey and Weinreb, 1993). Emanating from a central, inferior fibrous stalk, guinea pig laminar beams appear randomly oriented in cross- section, and are arranged in several layers.
Despite their architectural differences, laminar beams of both the rat and the guinea pig were readily identified in cross-sections of the optic nerve head. This approach allowed careful evaluation of the composition of individual beams, revealing important
RODENT L A M I N A CRIBROSA 133
FIG. 7. Horizontal longitudinal section of rat optic nerve head labeled for chondroitin-4 sulfate plus dermatan sulfate (A) demonstrates heavy label of laminar beams (between arrowheads), sclera (S), optic nerve septa and pia mater (P). Optic nerve labeling for chondroitin-4 sulfate alone (B) is restricted to the lamina cribrosa and sclera. (Original magnification, 95 × ).
interspecies similarities and differences (Table). Lami- nar composition of both species also showed numerous similarities to that previously reported in many studies on primates (Hernandez et al., 1986, 1987; Morrison et al., 1988b, 1994; Goldbaum et al., 1989; Caparas et al., 1991; Sawaguchi et al., 1992). This includes the presence and distribution of interstitial collagens, elastin and basement membrane components within individual laminar beams.
In both animals, labeling for chondroitin-4 sulfate plus dermatan sulfate produced results similar to the primate, with confluent label of the sclera, laminar beams and optic nerve septa and meninges (Morrison et al., 1994), indicating that proteoglycans containing
FIG. 8. Horizontal section of guinea pig optic nerve head labeled for chondroitin-4 sulfate plus dermatan sulfate (A) shows heavy label of laminar beams (between arrowheads), sclera (S) and optic nerve septa. Labeling for chondroitin-4 sulfate alone (B) results in a virtually unstained lamina and retrobulbar optic nerve (Original magnification, 95 x ).
these glycosaminoglycans appear to be present in optic nerve head connective tissues in all animals studied to date.
However, labeling patterns for chondroitin-4 sulfate proteoglycans alone indicated a more restricted dis- tribution with a possibly significant difference between rats and guinea pigs. In the rat, antibody labeling for chondroitin-4 sulfate containing proteoglycans was limited to the sclera and the transition zone of the optic nerve head, a pattern identical to that observed in both humans and monkeys (Morrison et al., 1994). This supports our contention that these structures are equivalent to rat lamina cribrosa. Apparently, in rats, as well as primates, unique chondroitin-4 sulfate- containing proteoglycans reside within the lamina
134 J. M O R R I S O N ET AL.
IL
FIG. 9. Rat and guinea pig optic nerve heads labeled with antibodies to chondroitin-6 sulfate show confluent label (arrows) of rat laminar beams (A). In the guinea pig (B), there is dense linear label along the margins of the beams (arrows), as well as around blood vessels within the beams (arrowhead). (Original magnification, 370 × ).
cribrosa and inner sclera, possibly in response to shearing forces generated by normal intraocular pressure. In contrast, chondroitin-4 sulfate proteo- glycan labeling in the guinea pig was absent from the lamina cribrosa and limited to the sclera, representing a significant departure from results previously reported in the primate.
While the laminar beams of both rat and guinea pig labeled with antibodies to chondroitin-6 sulfate proteo- glycan, the distribution of label throughout rat laminar beams resembled more closely the inter- mittent labeling of laminar beam margins with filamentary internal distribution recently seen in the h u m a n and rhesus lamina (Morrison et al., 1994). The guinea pig laminar beams clearly indicated a
restriction of this glycosaminoglycan to laminar basement membrane proteoglycans along beam mar- gins and blood vessels.
The potential roles of all of these extracellular matrix components in governing lamina cribrosa function has already generated extensive speculation in similar studies in primate tissue and is beyond the scope of the current discussion. While many of the ECM components demonstrated here are primarily structural and stable, the ECM as whole is a highly labile tissue whose response to stress depends heavily on functional proteins, many of which are cell- associated, easily denatured and highly susceptible to autolysis (Tervo et al., 1992; Rutka et al., 1988: Engle, 1991). Therefore, experiments designed to understand the composition and function of the lamina cribrosa ECM require strict control of ex- perimental conditions, fixation and tissue handling, all of which would be facilitated by routine use of these small, inexpensive animals.
These initial studies of the architecture and com- position of the lamina cribrosa in both rats and guinea pigs have revealed many similarities to the primate. These inexpensive, readily available laboratory ani- mals may prove appropriate models for studying the ECM of the optic nerve head to understand how its components govern the behavior of the lamina cribrosa and ultimately determine its relationship to axonal health and disease.
Acknowledgements
Supported by NIH grant EY10145 (Dr Morrison) and unrestricted funds from Research to Prevent Blindness. Dr Morrison is a 1990 RPB Miriam and Benedict Wolfe Scholar. Proprietary interest category: N.
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