effects of fibrinolysis on neurite growth from dorsal root ganglia cultured in two- and...

THE JOURNAL OF COMPARATIVE NEUROLOGY 365:380-391 (1996)

Effects of Fibrinolysis on Neurite Growth From Dorsal Root Ganglia Cultured in

Two- and Three-Dimensional Fibrin Gels

CURTIS B. HERBERT, GEORGE D. BITTNER AND JEFFREY A. HUBBELL Department of Chemical Engineering (C.B.H., J.A.H.) and Zoology (G.D.B.) and Institute for

Neuroscience (G.D.B., J.A.H.), University of Texas, Austin, Texas 78712

ABSTRACT The mechanism of neurite penetration of three-dimensional fibrin matrices was investi-

gated by culturing embryonic chick dorsal root ganglia (DRGs) within, fibrin gels, upon fibrin gels, and upon laminin. The length of neurites within three-dimensional matrices of fibrin was decreased in a concentration-dependent manner by agents that inhibited plasmin, e.g., aprotinin, or that inhibited plasminogen activation, e.g., E-aminocaproic acid (EACA), or plasminogen antiserum. In contrast, such agents in,creased the length of neurites growing out from DRGs cultured upon two-dimensional substrates of fibrin and had no effect on the length of neurites growing out from DRGs cultured upon laminin. Visualization of neurites within three-dimensional fibrin matrices demonstrated that the distance between fibrin strands was much smaller than the diameter of neurites. All these data were consistent with the hypothesis that fibrinolysis localized to the region of the neurite tip is an important mechanism for neurite penetration of a physical barrier of fibrin strands arranged in a three-dimensional matrix. c 1996 Wiley-Liss, h e .

Indexing terms: peripheral nerve regeneration, fibrin glue, plasmin, aprotinin, earninocaproic acid

Severed peripheral nerves are typically repaired by suturing together the epineurium or the perineurial fascicles of the two cut ends. In cases where the ends cannot be reapposed without exerting undue tension, severed nerves are often repaired by suturing an autograft between them (Idusskin and Battista, 1986; Mackinnon, 1989). Autograft- ing requires the removal of a functional nerve which may not even be available if the nerve gap is too long (Mackin- non, 1989). As an alternative to an autograft, a nerve guide tube may be used to repair a severed nerve. The nerve stumps are sutured into either end of the tube. leaving a gap between the ends of the stumps. The tube guides the regenerating axons and glia from the proximal to the distal stump and excludes cells from surrounding tissues. Func- tional recovery is often poor when severed peripheral nerves are repared with autografts or nerve guides (Lund- borg, 1990; Seckel, 1990).

A better understanding of the role and function of the naturally occurring fibrin matrix in peripheral nerve regeneration could suggest procedures to improve axonal regeneration through nerve guides. That is, when nerve guides are used to repair a 10-mm gap in the rat sciatic nerve, fibrin forms inside the tube between the stumps by 2-7 days post-severance (Williams et al., 1983; Williams 1987; Aebischer et a]., 1990). The regenerating axons and associated glia invade and grow through the fibrin; in the absence

of the fibrin, no nerve regeneration occurs. The inner diameter and microgeometry of the nerve guide influences the size of the naturally forming fibrin matrix and number of neurites that successfully traverse a gap in the rat sciatic nerve (Williams and Varon, 1985; Aebischer et al., 1990). The number of neurites that traverse the gap can be increased by adding fibrinogen-containing blood plasma, which changes the structure of the fibrin in the nerve gap (Williams, 1987; Williams et al., 1987). Finally, the addition of plasminogen affects the invasion of fibrin matrices by melanoma cells (Meissauer et al., 1992) and the migration of fibroblasts within fibrin clots (Knox et al., 1987).

In this study, we employed inhibitors of fibrinolysis to investigate the effect of fibrin degradation on neurite growth from cultures of embryonic chick dorsal root ganglia (DRGs) within fibrin, upon fibrin, and upon laminin.’ We also employed electron microscopy to compare the size of the spaces between fibrin strands to the size of neurites growing within fibrin gels. Our results indicate that neurit,e

Accepted July 21, 1995. JelTi-ey A. Hubbell, Ph.D., is now at Division of Chemistry and Chemical

Engineering, Mail code 21041, California Institute of Technology, Pasa- dena, CA 91125. Address reprint requests there.

‘In this paper, “upon” refers to a two-dimensional process (e.g., neurites growing upon a fibrin surface), whereas “within” refers to a three- dimensional process (e.g., neurites growing within a fibrin matrix).

c 1996 WILEY-LISS, INC.

NEURITE GROWTH WITHIN FIBRIN

Factor XIIIa i

381

Aprotinin Fibrin Degradation

Plasmin Products

Fibrinogen

Thrombin

Fibrin 1 I Crosslinked

Fibrin

Activators

Plasminogen

Fig. 1. Schematic description of fibrin polymerization and proteolysis. Thrombin polymerizes fibrinogen into fibrin which is degraded by plasmin. Activation of plasminogen into plasmin is inhibited by anti- plasminogen antiserum and t-aminocaproic acid (EACA). Aprotinin inhibits fibrinolysis by plasmin. Inhibitors of the plasmin cascade are italicized.

growth within fibrin, but not upon fibrin or upon laminin, is facilitated by local fibrinolysis which permits neurites to create a path for growth within fibrin. Our results suggest that fibrin density and structure should affect peripheral nerve regeneration through the fibrin bridge that spontaneously forms within nerve growth guides.

MATERIALS AND METHODS Figure 1 schematically describes the role of various

substances involved in fibrin polymerization and proteolysis. Human fibrinogen containing only trace amounts of plasmin and plasminogen2 was obtained from Sigma Chemi- cal (St. Louis, MO). Bovine thrombin, aprotinin (a serine protease inhibitor that inhibits plasmin), €-amino caproic acid (EACA, an inhibitor of plasminogen activation), goat antiserum against human plasminogen, goat antiserum against human immunoglobulin A (IgA), goat antiserum against rabbit immunoglobulin G (IgG), insulin-transferrin- sodium selenite medium supplement, type 7s nerve growth factor (NGF), antibiotic-antimycotic mixture, bovine serum albumin (protease-free), and laminin were also purchased from Sigma. Culture medium constituents such as Ham's F12 nutrient mixture and Hanks' buffered saline solution (HBSS) were obtained from GIBCO (Grand Island, NY). Fertile White Leghorn chicken eggs were obtained from SPAFAS (Norwich, CT). Tris-buffered saline (TBS) was made from 25 mM Trizma base (Sigma), 2.7 mM KC1, 138 mM NaCl, and adjusted immediately before use to pH 7.4 at 37°C.

Dissection and culture medium Dorsal root ganglia were dissected from 7-day-old chick

embryos (Varon et al., 1972) and stored in HBSS at 4°C for 1-4 hours. DRG culture medium was a modified N1 medium consisting of insulin (5 pgiml), transferrin (5 pg/ml), sodium selenite (5 ng/ml), NGF (50 ng/ml), 0.2%

W.0004 Sigma units plasmin and 0.008 Sigmaunits plasminogenper mgof fibrinogen, respectively; each Sigma unit approximately equals 3 World Health Organization units.

bovine serum albumin, and 1% antibiotic-antimycotic mixture in Ham's F12 (Bottenstein, 1980; Bottenstein et d., 1980; Skaper et al., 1982). The culture medium was filter- sterilized with a 0.2-pm syringe filter. After being placed within fibrin, upon fibrin, or upon laminin, DRGs were incubated in culture medium at 37"C, 5% COZ, and 100% relative humidity.

Culture upon laminin Laminin was diluted to a concentration of 100 pgiml in

sterile 50 mM sodium carbonate buffer (pH 9.0). Glass coverslips were soaked in 2 M NaOH for 2 hours, thoroughly rinsed in deionized water, and autoclaved. The coverslips were rinsed twice in sterile carbonate buffer, covered with 25 p1 of laminin solution, and incubated for 4 hours at 37°C in 100% relative humidity. The coverslips were rinsed twice in sterile TBS and transferred into a 24-well plate containing 1 mliwell of culture medium. DRGs were then placed one per well upon the laminin- treated coverslips and centered in the well with a pipette tip.

Culture upon and within fibrin Fibrinogen (9.6 mgiml) was dissolved in 10 ml TBS and

the original buffer salts were removed by dialysis against 3 liters of TBS for 2 days at 4°C. The fibrinogen solution was filter-sterilized with 0.45-pm syringe filters and stored at 4°C for up to 1 week. Thrombin was dissolved in TBS and stored in aliquots (1,000 NIH unitsiml) at -20°C for as long as 1 week; thrombin solutions were thawed only once. Immediately before use, thrombin was thawed and diluted with TBS containing 20 mM CaClz substituted for 30 mM NaCl. For culture within fibrin gels, 50 pl of the fibrinogen solution was put on a sterile petri dish, combined with 10 pl of HBSS containing one DRG, and mixed with 60 cl.1 of the thrombin solution. The final fibrin composition was calculated to be 4 mgiml fibrinogen, 2 NIH units/ml thrombin, and 10 mM CaC12. Before it became a solid gel, 100 ~1 of the mixture was transferred into a well of a 24-well culture plate in a drop that was not allowed to touch the sides of the well. A pipette tip was used to center the DRG in the well. After 30-60 minutes, 900 ~1 of culture medium was added. Culture upon fibrin gels was identical with that described for culture within fibrin gels, except that the DRG was added after the fibrinogen had formed a gel. Aprotinin and EACA were dissolved in TBS, filter-sterilized, and added in 10-p1 volumes to the medium to obtain the concentrations indicated, based on the total volume of medium plus fibrin in the well. The antisera were reconstituted in sterile deionized water so that the anti-plasminogen and anti-IgA antisera had a protein content of 32 and 37 mgiml, respectively. Both antisera were free of preservatives and were added in 30-p1 volumes to obtain a dilution of 33:l and final concentrations of 1 mgiml of anti-plasminogen antiserum and 1.1 mgiml of anti-IgA antiserum.

Fibrin gels that did not contain DRGs were made as described above and incubated in culture medium for as long as 3 months. After incubation, gel samples were transferred into a 10-ml glass vial containing 5 M urea in TBS (pH 7.4).

Electron microscopy Fibrin gels were visualized by scanning electron micros-

copy (SEM) to determine the size and spacing of fibrin strands. To minimize shrinkage of the fibrin gels in the

382

dehydration and critical point drying stages of preparation for SEM, 100 p1 of fibrin was made within 2-cm-long sections of sterile 5-mm inner diameter polystyrene serologi- cal pipettes. The gels were allowed to form at room tempera- ture for 30 minutes and were then immersed in TBS containing 10 pgiml aprotinin. After 24 hours at 37T, the gels were fixed at 4°C for 6 hours by immersion in TBS containing 2% glutaraldehyde (Polysciences; Warrington, PA). The gels were rinsed in three successive TBS baths and postfixed for 24 hours in 2% Os04 (Polysciences) dissolved in TBS. The gels were thoroughly rinsed in TBS, dehydrated in a gradually increasing gradient of ethanol in deionized water (2576, 3796, 50%, 63%, 7596, 90%, 95%, 2 x 100% for 30 minutes each), and critical point dried within the pipette, during which the gel shrank in volume by approximately 30%. The dehydrated gel was carefully removed from the pipette, mounted, sputter coated, visualized by SEM (model 515; Phillips Electronic Instruments, Mahway, NJ), and photomicrographed using PIN film (type 55; Polaroid Corp., Cambridge, MA).

The relative sizes of neurites and the spaces between fibrils in fibrin gels were investigated with transmission electron microscopy (TEM). The procedure used for making gels for SEM was repeated except that the fibrin contained ten DRGs and was incubated in DRG culture medium with no aprotinin for 48 hours. The gels were rinsed three times for 15 minutes each in 0.1 M cacodylate buffer (pH 7.41, fixed overnight at 4°C in a solution of 3% paraformalde- hyde, 3% glutaraldehyde, and 0.1% picric acid (w/w) in 0.1 M cacodylate buffer, and postfixed for 3 hours in 2% osmium tetroxide plus 1.5% potassium ferricyanide in 0.1 M cacodylate buffer. After dehydration in a gradually increasing gradient of ethanol in deionized water as described in the preceding paragraph, the gels were gently pushed out of the pipettes, embedded in Spurr’s resin (Polysciences; 50%, 6796, 100%, 100% Spurr’s in ethanol), and polymerized at 65°C overnight. Thin sections (75-nm nominal thickness) were prepared using a diamond knife and a Sorvall MTI ultramicrotome, placed on formvar- coated grids, and stained with lead citrate using Reynold’s lead. Electron microscopy was performed using a Siemens Elmiskope IA at 60 kV accelerating potential.

Measurement of neurite lengths DRGs within fibrin, upon fibrin, or upon laminin were

visualized with a high-resolution video camera (67 M series; Dage-MTI, Wabash, MI) attached to an inverted-stage microscope (Fluorovert; Wild Leitz, Rockleigh, NJ) equipped with a 2 . 5 ~ objective (Carl Zeiss, Thornwood, NY). The outlines of the cluster of cell bodies in the ganglion and the wave-front of the growing neurites were traced using a video monitor and an in-line image processor (Argus; Hamamatsu Photonic Systems, Bridgewater, NJ). The average neurite length was calculated as the width of an annulus of area equal to the area of the neurite zone, i.e., L = (~/TP~~[(A~,,,, + A,,,te,) - (Ainner)’~21, with Ainner = the area covered by the dense cluster of cell bodies in the ganglion, kUter = the area between the cluster of cell bodies and the leading edge of the wavefront of growing neurites, and L = the average neurite length for a circular ganglion in the center of a circular neurite wavefront.

Statistics Differences between samples and their controls were

reported as significant if they exceeded a 95% confidence

C.B. HERBERT ET AL.

limit as determined by a one-way analysis of variance and the Scheffe F post-hoc test.

RESULTS Fibrin gel structure

Fibrin gels were made as described in Materials and Methods. When the thrombin and fibrinogen solutions were combined, a firm gel was formed within 15 minutes. The gel was not perfectly clear, but was sufficiently translucent to permit light microscopic observation of both cell bodies and neurites. As a test for stability, some gels were made within the wells of culture plates, incubated in culture medium for 30 minutes or for 3 months, removed with a spatula, and transferred into glass vials containing 5 M urea. None of these gels dissolved within 60 minutes in 5 M urea, suggesting that the fibrin had been chemically crosslinked by small amounts of Factor XIII, which is a contaminant of fibrinogen (see also, e.g., Grinnell et al., 1980; Blomback and Okada, 1982).

Fibrin gels visualized with SEM (see Materials and Methods) consisted of long, densely packed strands crosslinked to each other at many points (Fig. 2A). The fibrin strands were randomly oriented and were typically less than 0.1 km in diameter.

Morphology of DRGs cultured within and upon fibrin and upon laminin

Neurites grew outward from DRGS cultured within three-dimensional fibrin gels, producing a roughly circular profile as viewed from above (Fig. 2B). Most neurites traveled downward as they grew out from the ganglion body, forming an angle of 15-60 degrees below the horizon- tal at a distance of 100-500 pm from the ganglion body. After 48 hours, the overall neurite pattern resembled an empty, inverted cone with a base much wider than its height, with the ganglion body at its apex and the neurites forming the walls. The neurites were suspended within the fibrin gel as determined by comparing the focal plane of the neurites and surface of the petri dish. Neurites tdypically grew in advance of migrating non-neuronal cells and were not fasciculated at their ends (Fig. 2C).

DRG cultures within fibrin were observed for indications that the DRGs caused degradation of the fibrin gel. During the first 2 days of culture within fibrin, the growing neurites did not move when the side of the culture plate was tapped. After 3-5 days, tapping the culture plate caused the neurites to move freely between their tips and the ganglion body. Shaking the culture plate caused the neurites to come loose at their tips and recoil in a manner suggesting that they were under tension and immobilized only at their tips. The neurite ends then floated freely as the culture plate was tapped, indicating that the fibrin adjacent to the DRG was degraded. After an additional 2 days, a transparent zone 2-3 mm in diameter formed in the fibrin around the DRG. At this time, the DRG was typically free-floating within the transparent zone, but the remainder of the gel was not visibly altered. The addition of a plasmin inhibitor (aprotinin, EACA, or anti-plasminogen antiserum) delayed the degradation of the gel around the DRG. For instance, the addition of 1 pgiml aprotinin caused a delay in the appear- ance of the transparent zone by 1-2 days, and 40 pgiml aprotinin caused a delay of 3-4 days.

Cultures of DRGs within fibrin were examined with TEM to determine the relative size of neurites (n = 50) compared

NEURITE GROWTH WITHIN FIBRIN 383

Fig. 2. Morphology of fibrin (A) and neurite outgrowth from DRGs entrapped within three-dimensional fibrin matrices (B, C). A SEM of fibrin structure. Scale har = 1 pm. B: Photomicrograph focused on ends of neurites (arrow) growing from a DRG (d) in a roughly circular

pattern. Scale bar = 500 km. C: Photomicrograph of neurites (arrows) growing in advance of migrating cells (arrowheads). Scale bar = 100 pin. B and C were photographed after 48 hours of culture within fibrin.

384

to the spaces between the fibrin strands (Fig. 3). Neurites were identified as such by the presence of microtubules, the absence of a nucleus, and the absence of rough endoplasmic reticulum. Growth cones were identified by the absence of all three of these elements and the presence of much smooth endoplasmic reticulum. Neurites in transverse section typically appeared as slightly irregular circles with a diameter of 1-1.5 pm. In longitudinal section, the diameter of a neurite often varied by 25-50% along its length (Fig 3A,C). Growth cones appeared as ellipsoids with diameters of 1.5-2.5 pm (Fig. 3B). Fibrin strands appeared as low- contrast rods in longitudinal section or as solid circles in transverse section with a diameter of 0.01-0.05 p m (Fig. 3A,C). The fibrin strands were typically separated by 0.1-0.5 pm. In all cases, neurites were substantially larger than the spaces between fibrin strands. The spacing of the strands was usually consistent throughout the gel; in some cases, fibrin strands were not observed within 1 pm of the neurite.

Our initial attempts to culture DRGs upon fibrin yielded variable results when no inhibitors were added to the gel. Of 20 DRGs cultured upon fibrin, eight did not attach to the fibrin and 12 appeared to be loosely attached but extended very few or no neurites. When 1 pg/ml aprotinin or 10 pglml EACA was added to the gel, DRGs consistently attached (n = 23/24 for aprotinin and 17118 for EACA) to the fibrin and extended neurites. For this reason, experiments involving culture of DRGs upon fibrin were conducted in the presence of 1 pg/ml aprotinin. Duplicate cultures including 1 yg/ml aprotinin within fibrin were similarly examined. In this way, it was possible to examine the dependence of neurite extension for EACA concentrations of 1-100 pg/ml upon fibrin as well as within fibrin.

When DRGs were cultured upon the surface of fibrin gels in the presence of 1 pg/ml aprotinin, neurites formed a fasciculated network of many connected branches (Fig. 4A,B). The neurites usually grew from all sides of the DRG at roughly the same rate, forming a starburst pattern (Fig. 4A). The neurites grew upon the fibrin and not within it, since the neurites and associated non-neuronal cells could be dislodged from the surface by gently scraping the fibrin with a pipette tip, uncovering an underlying layer of fibrin which could be peeled off the plastic culture plate. Non- neuronal cells (identified as such by their polygonal shape) were typically located just behind the growing tips of the neurites, i.e., non-neuronal cells did not migrate faster than the neurites extended. Non-neuronal cells were almost confluent close to the DRG, but were less numerous close to the tips of the neurites (Fig. 4B,C).

DRGs were also cultured upon laminin, which is much less subject to degradation by plasmin than is fibrin and is a useful reference material for studying how plasmin might directly affect neurite extension. DRGs cultured upon laminin-coated coverslips grew a dense network of intercon- nected neurites (Fig. 5A). After 48 hours, the neurites had formed fascicles near the body of the DRG, but remained unfasciculated near their distal ends. Glial cells as well as a few round, phase-bright cells that were either neuronal cells or mitotic glial cells were present in the region between the DRG body and halfway to the neurite tips (Fig. 5B). Almost no cells were present in the region covered by the outermost third of the neurites (Fig. 5C).

C.B. HERBERT ET AL.

Fig. 3. TEM of neurites growing within a three-dimensional fibrin matrix. A. Oblique section of a neurite. Arrow indicates a microtubule. Arrowheads by (0 in A and C indicate fibrin strands in longitudinal/ oblique section. B: Growth cone with abundant smooth endoplasmic reticulum (arrows). C: Neurites with areas rich in transversely sec- tioned microtubules (m). Scale bar = 0.5 K r n for A-C.

Effect of protease inhibitors on neurite extension

We used several protease inhibitors (aprotinin, EACA, and anti-plasminogen antiserum) to determine if proteolytic activity was necessary or important for the growth of neurites within three-dimensional fibrin matrices. Aproti- nin caused a concentration-dependent reduction of neurite extension within fibrin (Fig. 6A). Neurites grew out from the DRG in the absence of aprotinin; neurite length was progressively reduced as the concentration of aprotinin was increased from 1 to 40 pg/ml. For example, 1 pg/ml aprotinin significantly (P < .05, see Materials and Meth- ods) reduced neurite extension by 28%' within fibrin matrices compared to cultures with no aprotinin, and 40 pgirnl


Fig. 4. Morphology of neurite outgrowth from DRGs cultured for 48 hours upon two-dimensionai fibrin matrices in the presence of 1 pg!ml aprotinin. A: Photomicrograph of neurites (arrows1 near the DRG cd). n, Non-neuronal cells. Scale bar = 500 pm. B: Photomicrograph of a region corresponding to that of n shown in A above, showing neurites

(arrow) with non-neuronal cells (arrowhead). Focus is on the center of the field of view. Scale bar = 100 Fm. C: Photomicrograph of a region more distal to n, showing neurites (arrow) and non-neuronal cells (arrowhead). Note that there are fewer non neuronal cells than in B. Scale bar 7 100 pm.

386 C.B. HERBERT ET AL.

Fig. 5. Morphology of neurite outgrowth from DRGs cultured for 48 hours upon laminin. A: Photomicrograph of neurites growing from a DRG (d). Scale bar = 500 p,m. B: Photomicrograph of an area approximately 100 Fm from the DRG, showing cell bodies (arrows) and a dense

network of neurites. Scale bar = 100 krn. C: Photomicrograph taken more distal to iB) near the growing ends of the neurites, showing the branched pattern of neurite growth and an absence of cell bodies. Scale har = 100 p,m.


aprotinin significantly reduced neurite extension by 70%. In contrast, aprotinin was necessary for neurite growth upon fibrin, since DRGs detached from two-dimensional fibrin substrates and no neurite growth was observed in the absence of aprotinin. At 40 pg/ml, aprotinin caused a significant increase in neurite length (28%) upon fibrin compared to cultures with 1 pg/ml aprotinin (Fig. 6C). Compared to I pgiml, 5-20 pgiml aprotinin had no significant effect. Upon laminin, aprotinin (up to 40 pg/ml) had no effect on the length of neurites growing from DRGs compared to cultures with no aprotinin (Fig. 6E). These results demonstrate that addition of aprotinin reduced neurite extension within fibrin but not upon fibrin or upon laminin.

Since aprotinin can block not only plasmin but also other serine proteases such as trypsin, elastase, and possibly urokinase plasminogen activator (uPA) (Fritz and Wun- derer, 19831, we tested the effect of a more selective inhibitor of plasmin (EACA) on neurite growth. This inhibitor is a lysine analog that competitively inhibits the attachment of plasmin and plasminogen to fibrin (Mosher, 1990). Compared to control cultures growing within fibrin lacking aprotinin or EACA, the addition of EACA reduced neurite length within fibrin as follows: 8% at 1 pgiml, 22% at 10 pgiml, and 77% at 100 pg/ml (n = 18-24 DRGs at each concentration). The reduction by 10 and 100 pg/ml EACA was significant compared to control cultures. In contrast, DRGs detached from the surface and did not consistently grow neurites upon fibrin unless 10 pg/ml of EACA was present. Upon laminin, 1, 10, or 100 pg/ml EACA had no significant effect on neurite length compared to control cultures with no EACA (n = 18-24 DRGs at each concentration).

Since DRGs detached from fibrin surfaces when only 1 pg/ml EACA was present, cultures within and upon fibrin were performed with varying concentrations of EACA (1-100 kg/ml) in the presence of 1 pgirnl aprotinin. Cultures were also performed upon laminin in the presence of 1 kg/ml aprotinin with varying concentrations of EACA. When we cultured DRGs within fibrin in the presence of 1-100 kg/ml EACA plus 1 pgiml aprotinin, EACA plus 1 pg/ml aprotinin significantly reduced neurite length by 46%) at 10 pgiml and by 56% at 100 pg/ml, but had no significant effect at 1 pgiml compared to cultures containing only 1 pg/ml aprotinin (Fig. 6B). The trend in this result was similar to that observed for EACA alone (see Results above). Upon fibrin, EACA plus 1 pg/rnl aprotinin significantly increased neurite length by 43%' at 10 pg/ml EACA and by 51% at 100 pgiml, but had no significant effects at 1 pgiml compared to cultures with 1 pg/ml aprotinin alone (Fig. 6D). Upon laminin, EACA (1-100 pg/ml) plus 1 pgirnl aprotinin had no significant effect on the length of neurites compared to control cultures containing only 1 pgiml aprotinin (Fig. 6F) or to cultures containing only EACA. This lack of effect was also observed for cultures with EACA only. These results showed that EACA and/or aprotinin reduced neurite lengths within fibrin in a concentration-dependent manner, increased neurite lengths upon fibrin, and had no effect on neurite lengths upon laminin.

Finally, we specifically blocked plasminogen activation in DRG cultures with a polyclonal antiserum against plasminogen (Figs. 7, 8). Addition of anti-plasminogen antiserum reduced the average neurite length within fibrin by 68% compared to addition of a control antiserum against already

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Fig. 6. The effect of aprotinin (A,C,E) or EACA plus 1 pg/ml aprotinin (B,D,F) on neurite length within three-dimensional fibrin matrices (A,B), upon two-dimensional fibrin matrices (C,D), or upon laminin (E,F). All neurite lengths are normalized relative to control cultures shown in the first bar of each graph. Control cultures were performed with no aprotinin (A,C,E) or with 1 pgiml aprotinin and no EACA (B,D,F). Each bar represents an average neurite length 2 SEM of 19-24 DRGs cultured for 48 hours. The average neurite length of the controls in A, C, and E, is 658, 853, and 1,828 hm, respectively. The average neurite length of'the controls in B, D, and F, is 447, 697, and 1,870 pm, respectively. ksterisks indicate normalized neurite lengths significantly different (P < ,051 from the control. An increase in aprotinin concentration reduces neurite length within fibrin (A) but increases neurite length upon fibrin (C). Aprotinin did not affect the length of neurites upon laminin (El. As EACA concentration increases in the presence of 1 pg/rnl of aprotinin, neurite length within fibrin is reduced (B), but neurite length upon fibrin is increased (D), and neurite length upon laminin is not affected (F).

defined IgA (Fig. 7A). When anti-plasminogen antiserum was added in the presence of additional 1 pg/ml aprotinin, neurite length within fibrin was reduced by 63% compared to cultures with the control antiserum plus 1 pgirnl aprotinin (Fig. 7B). In contrast, the anti-plasminogen antiserum plus 1 pg/ml aprotinin did not affect the length of neurites upon fibrin (Fig. 7C). These results showed that addition of anti-plasminogen antiserum reduced neurite growth within fibrin, but not upon fibrin. The goat anti- human plasminogen antiserum and the control antiserum, goat anti-human IgA, were used as received and were not


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Fig. 7. The effect of anti-plasminogen antiserum on neurite length within three-dimensional fibrin matrices (A,B) or upon two-dimensional fibrin matrices (C) without aprotinin (A) or with 1 pg/ml aprotinin (B,C). Neurite length is normalized relative to cultures containingcontrol antiserum shown in the first bar of each graph. Each bar represents an average neurite length ? SEM of 19-24 DRGs cultured for 48 hours. The average neurite lenglh of the controls for A, B, and C is 718, 484, and 878 pm, respectively. Asterisks indicate neurite lengths significantly different (P < .051 from the control cultures. Anti-plasminogen antiserum reduced neurite lengths wilhin fibrin in the absence of aprotinin (A) or in the presence of 1 kg/ml aprotinin (B). Anti-plasminogen antiserum plus 1 pgiml aprotinin had no measurable effect on neurite lengths upon fibrin (GI.

tested for the presence of plasmin activators or inhibitors. For this reason, we conducted experiments on DRGs cultured within fibrin with a second control, goat anti-rabbit IgG (n = 12; data not shown). This control did not inhibit neurite growth. All three antisera were obtained from the same source and were prepared by fractionation and ion- exchange chromatography.

DISCUSSION A better understanding of the factors that affect neurite

extension within three-dimensional matrices could help in the design of new materials and in the modification of naturally occurring materials used for peripheral nerve repair. The investigation of neurite growth within fibrin is of special interest because fibrin has been shown to support sciatic nerve regeneration (Williams et al., 1983; Aebischer et al., 19901. Three-dimensional fibrin matrices have also been shown to support the growth of neurites from explants of rabbit retinal ganglia in culture (Sonnentag et al., 1992). Kone of these studies have examined the effect of protease inhibitors on neurites growing into a two- or three- dimensional fibrin matrix.

The mechanism of axonal outgrowth within three- dimensional matrices such as fibrin has not been clearly established (Seeds et al., 1992a). One hypothesis is that the proteases at or near the neurite tip degrade the matrix strands to create a pathway for growing neurites (Krysto- sek and Seeds, 1978; Seeds et al., 1992a). A second hypothesis is that axonal growth cones physically push aside the matrix strands (Cajal, 1890). A third hypothesis is that axons weave a path between the strands of the fibrin matrix.

We investigated the growth of neurites within fibrin by 1) measuring the effect of protease inhibitors on the length of neurites growing from DRG explants cultured upon laminin and within and upon fibrin made with 4 mg/ml of fibrinogen, a concentration similar to that in blood clots (Mosher, 1990) and in the spontaneously forming fibrin bridge that connects sciatic nerve stumps that have been sutured into silicone nerve growth guides (Williams, 1987), and by 2) comparing the size and spacing of fibrin strands in the gels to the size of neurites growing within them. Three protease inhibitors were used (aprotinin, EACA, and polyclonal anti-plasminogen antiserum) that act by different mechanisms: Aprotinin blocks the active site of plasmin, EACA blocks the lysine-binding sites of plasminiplasminogen, and the antiserum probably blocks various epitopes on plasminiplasminogen. Any one of the three inhibitors tested could have exerted an effect by some mechanism besides blocking plasmin/plasminogen: aprotinin by blocking some other serine protease such as trypsin, EACA by blocking the lysine-binding sites of an unknown substance, or the antiserum by blocking some unknown plasminogen- like protein. It is very likely that they all acted by inhibiting plasminiplasminogen since all three inhibitors affected neurite outgrowth in a similar manner.

Neurite growth within fibrin Experiments with fibrinolysis inhibitors indicated that

fibrinolysis was necessary for neurites to grow within fibrin gels. The plasmin inhibitor aprotinin and the plasminogen activation inhibitor EACA both reduced neurite growth in a concentration-dependent manner within fibrin. Anti-


plasminogen antiserum also inhibited neurite growth within fibrin.

Transmission and scanning electron microscope observa- tions suggested that neurites were too large to weave a path through the fibrin strands of the gel matrix without a mechanism for removing or pushing aside the fibrin strands. For example, TEM data showed that neurites growing within fibrin were typically 1-1.5 pm in diameter, whereas the spaces between the fibrin strands were about 0.1-0.5 Fm wide. That is, all neurites were surrounded by a fibrin matrix with strands too closely packed for the neurites to pass freely through the spaces between them, even after accounting for shrinkage (approximately 30% in volume) of the fibrin matrix during dehydration. Furthermore, our SEM data showed that most fibrin strands were many microns long (see Fig, 2A), making it unlikely that neurites grew around the ends of the fibrin strands.

If neurites cannot grow between or around fibrin strands, then the neurites must push the strands aside or degrade them. We obtained no TEM data suggesting that the outgrowing neurites distort the fibrin matrix as might be expected if fibrin strands were pushed aside. That is, fibrin containing DRGs had a similar strand size and spacing both adjacent to, and far from, the neurites and growth cones. These data also suggest that any fibrinolysis occurred in a region close to the growth cone, similar to that reported for sensory neurons which release urokinase plasminogen activator (uPA) at the neurite tip (Seeds et al., 1992a). In some cases, fibrin was removed from a region less than 1 Fm around the neurite. We obtained no TEM data suggesting that outgrowing neurites degraded fibrin for several microns around the growth cone to create large gaps between the fibrin strands. In contrast, Seeds et al. (1992b) observed evidence of fibrin degradation for several microns around the growth cone of a sensory neuron overlayed with fibrin. It may be that such a widespread fibrinolysis was not observed around the neurites in our cultures because our fibrinogen contained lower concentrations of plasmin and plasminogen compared to the fibrinogen used by Seeds et al. (1992b). Since glial cells migrating within fibrin lagged behind the leading neurites in our experiments, it seems likely that the neurite tips degraded fibrin without direct assistance from glial cells. We did observe widespread fibrinolysis near the DRG. This fibrinolysis could have been caused by tissue plasminogen activator (tPA) released by Schwann cells (Baron-Van Evercooren et al., 1987) and/or by uPA released from the neuronal somas which were present in the DRG (Seeds et al., 1992bj.

Proteolysis has been shown to be important for neurite growth within other three-dimensional matrices that did not contain fibrin. For example, aprotinin or a cocktail of EACA plus soybean trypsin inhibitor reduced the length of chick DRG neurites growing within three-dimensional astrocyte cultures, but had no effect on neurites extending upon astrocyte monolayers or upon collagen (Fawcett and Hous- den, 1990). Similarly, inhibitors of collagenase blocked neurite extension within, but not upon, collagen gels (Pitt- man and Williams, 1988; Pittman et al., 1991).

Neurite growth upon fibrin or laminin Plasmin or plasminogen may affect neurite lengths via

second messengers in other culture systems (Baron-Van Evercooren et al., 1987; Kalderon, 1990; Pittman et al., 1991). If plasmin or plasminogen directly or indirectly activated second messengers that decreased neurite growth

from DRGs cultured within fibrin, then plasmin/plasminogen inhibitors should have decreased neurite growth upon fibrin as they did within fibrin. In contrast, such inhibitors (aprotinin, EACA) increased, rather than decreased, neurite growth upon fibrin. In fact, in the absence of aprotinin or EACA, DRGs often did not even attach to the gel or grow neurites. Aprotinin and EACA had no effect on neurite growth when DRGs were cultured upon laminin, further indicating that these substances had no effect on neurite length independent of fibrinolysis inhibition.

The increase in neurite length upon fibrin caused by the addition of protease inhibitors could result from a change in the balance of proteases and protease inhibitors at the neurite tip. The hypothesis that a balance of proteolytic activity is necessary for optimal neurite growth (Hawkins and Seeds, 1986; Monard, 1988) is consistent with observa- tions that the addition of protease inhibitors, e.g., soybean trypsin inhibitor, to in vitro cultures of neuronal cells or ganglia can increase neurite length at a low concentration and inhibit neurite length at a high concentration (Hawk- ins and Seeds, 1989; Pittman et al., 1989). In our experiments, the protease inhibitors might have shifted the protease inhibitor-protease balance so that the fibrin underlying points of neuritic attachment was protected from degradation, causing a stabilization of these points and a resultant increase of neurite length.

Implications of these studies Our data suggest that proteolytic activity immediately at

the tip permitted the growth of chick DRG neurites within fibrin gels. These results are consistent with the hypothesis that the barrier of fibrin strands must be degraded by outgrowing neurites to create an opening between fibrin strands (Krystosek and Seeds, 1978; Seeds et al., 1992a). Our studies suggest that a three-dimensional culture system is necessary to model neurite growth in vivo. Indeed, the effects of fibrinolysis inhibitors on neurite growth within fibrin were the opposite of the effects upon fibrin.

Our results also have implications for the therapeutic use of fibrin in glues used to reconnect severed peripheral nerves. For example, the concentration of aprotinin in fibrin glues varies in the formulations available for clinical use, but aprotinin typically exceeds concentrations that strongly inhibit neurite extension within fibrin in our study. For example, in our study 20 pg/ml aprotinin reduced neurite extension by 50% (ICbO). In contrast, Tissucol’E fibrin glue (Immuno AG) contains a final concentration of aprotinin equivalent to approximately 330 pgiml of the aprotinin used in our study, and Biocoll” (Centre de Transfusion Sanguine de Lille) contains an aprotinin concentration equivalent to about 2,200 pgiml (Sierra, 1993). Since these glues contain various concentrations of plasminogen, the ICS0 in a clinical setting may be different than that found in our in vitro system. Nevertheless, our results suggest that the high concentration of plasmin inhibitors used in these glues could actually reduce neurite outgrowth, especially if the glue were to be introduced between severed nerve ends.

ACKNOWLEDGMENTS The authors are indebted to Robert Lewis for performing

the many chick embryo dissections necessary for this investigation. We thank Paul Letourneau for demonstrat- ing a chick embryo dissection. We also thank John Weisel

NEURITE GROWTH WITHIN FIBRIN

for advice on SEM visualization of fibrin gels and Noel Lucky and Arisa Sunio for technical assistance. SEM was performed using the facilities at the Cell Research Insti- tute. TEM was performed by Martis Ballinger. Funding was provided by the National Science Foundation to J.A.H. (ECS-8915178) and by the Texas Advanced Technology Project to G.D.B.

391

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effects of fibrinolysis on neurite growth from dorsal root ganglia cultured in two- and...

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