Injury-induced gelatinase and thrombin-like activities inregenerating and nonregenerating nervous systems

IGOR FRIEDMANN, ANAT FABER-ELMAN, ETI YOLES, AND MICHAL SCHWARTZ1

Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel

ABSTRACT It is now widely accepted that injurednerves, like any other injured tissue, need assistancefrom their extracellular milieu in order to heal. Wecompared the postinjury activities of thrombin andgelatinases, two types of proteolytic activities knownto be critically involved in tissue healing, in nonre-generative (rat optic nerve) and regenerative (fishoptic nerve and rat sciatic nerve) neural tissue.Unlike gelatinases, whose induction pattern was com-parable in all three nerves, thrombin-like activitydiffered clearly between regenerating and nonregen-erating nervous systems. Postinjury levels of thislatter activity seem to dictate whether it will displaybeneficial or detrimental effects on the capacity ofthe tissue for repair. The results of this study furtherhighlight the fact that tissue repair and nerve regen-eration are closely linked and that substances thatare not unique to the nervous system, but participatein wound healing in general, are also crucial forregeneration or its failure in the nervous system.—Friedmann, I., Faber-Elman, A., Yoles, E., Schwartz,M. Injury-induced gelatinase and thrombin-like activ-ities in regenerating and nonregenerating nervoussystems. FASEB J. 13, 533–543 (1999)

Key Words: thrombin z matrix metalloproteases z CNS z PNS

Neurons in the mammalian central nervous system(CNS)2 are unable to regenerate after axonal injury,whereas both the mammalian peripheral nervoussystem (PNS) and the CNS of lower vertebrates arecapable of regeneration (1). PNS axons fail to elon-gate more than 1 mm into optic nerve grafts (2). Incontrast, neurons from most regions of the mamma-lian CNS can elongate their axons into PNS grafts,presumably aided by the supportive cellular environ-ment of the peripheral nerve (3). It therefore seemsthat CNS neurons possess, at least in part, thepotential to regenerate, but lack the supportive andpermissive environment that characterizes regenera-tive systems after injury. Our research group recentlyproposed that the processes of tissue repair andregeneration in the nervous system have features incommon with those of any recovering tissue (4, 5).Injury of any tissue triggers a complex cascade ofevents that modifies the environment and induce a

process of tissue repair. In effecting these changes,molecular components such as proteases play acritical role (6–8). Failure of the CNS to regenerateafter injury might be the result of impairment of thetissue repair mechanism at one or more stages of thehealing cascade, including those involving proteaseparticipation.

One of the proteases known to influence thenervous system is thrombin, a key enzyme in bloodcoagulation (9). Thrombin acts as a potent mitogenthat modulates the morphology of astrocytes (10, 11)and can induce the secretion of nerve growth factor(NGF) by astrocytes (12). It inactivates acidic fibro-blast growth factor, known to promote neuritic out-growth and astrocyte proliferation (13–15). It inhib-its outgrowth from neuritic neuronal cells due tointeraction with the thrombin receptor PAR-1 (16,17), which is proteolytically activated by thrombin(7). Thrombin at high concentrations was shown toinduce apoptotic cell death in astrocytes and neu-rons cultured under normal conditions, whereas atmoderate concentrations it protected those cellsfrom a variety of metabolic insults (18–20). Botheffects were shown to be due to the activation of thePAR-1 receptor. The processes of neuroprotectionand apoptotic cell death display similar characteris-tics in the signal transduction pathways (19, 21, 22),and it was suggested that different concentrations ofthrombin might result in different activation levels ofthe same pathway (21).

Another group of proteases involved in woundhealing is the family of matrix metalloproteases(MMPs, collagenases). Two of these, gelatinase A(MMP-2, 72 kDa) and gelatinase B (MMP-9, 92 kDa),belong to the subfamily of gelatinases. MMPs arelargely responsible for the degradation of extracellularmatrix (ECM) components such as collagen and pro-

1 Correspondence: E-mail: bnschwar@weizmann.weizmann.ac.il2 Abbreviations: CNS, central nervous system; DMEM, Dul-

becco’s modified Eagle’s medium; ECM, extracellular matrix;IL, interleukin; MMP, matrix metalloprotease; MMP-2, gela-tinase A; MMP-9, gelatinase B; NGF, nerve growth factor; OD,optical density; PA, plasminogen activator; PN-I, glial-derivedprotease nexin I; PNS, peripheral nervous system; SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TIMPs, tissue inhibitors of matrix metalloprotease; TNF,tumor necrosis factor.

5330892-6638/99/0013-0533/$02.25 © FASEB

teoglycans in several normal and pathological pro-cesses, including tissue remodeling (23), and MMPgenes are among the most abundant of those ex-pressed by cells in inflammatory lesions (24). Remod-eling of the ECM by proteolytic activity is known to becrucial for growth-cone motility (8). Whereas thrombinis known to produce the matrix component fibrin (9,25, 26), MMPs are responsible for matrix degradation.Fibrin fibronectin-containing matrix promotes thegrowth of cultured septal and hippocampal neuronsfrom newborn rats (27). The MMPs are activated by thefibrin-degrading plasmin, which is itself activated byplasminogen activators (PA) (24). Cultured neuronsfrom the PNS release at least two proteases capable ofdegrading ECM components, a calcium-dependentMMP and a PA (28). In addition, NGF-induced expres-sion of gelatinase A by dorsal root ganglionic neuronsoccurs in correlation with the ability to degrade ECMand to extend neurites (29), and NGF production inastrocytes is induced not only by thrombin but also bygelatinases (12). In contrast to the above findings,which point to beneficial effects of MMPs in thenervous system, several studies hint at the involvementof MMPs in processes detrimental to the brain. Intra-cerebral injection of bacterial collagenase as well as oftumor necrosis factor (TNF) -a induces gelatinase Bproduction, which causes delayed opening of theblood–brain barrier (30, 31). Patients with inflamma-tory neurological disorders, including multiple sclero-sis, show increased gelatinase B in the cerebrospinalfluid (32). Gelatinase B is also present in the cerebro-spinal fluid of mice with experimental allergic enceph-alomyelitis, the animal model of multiple sclerosis,where it cleaves myelin basic protein (33).

To investigate the relationship of gelatinase andthrombin activities to regenerative and degenerativeprocesses in the nervous system in vivo, we comparedtheir activities in response to axonal injury in tworegenerative nervous systems (rat sciatic nerve andfish optic nerve) with a nonregenerative nervoussystem (rat optic nerve). We show that the inductionof gelatinase activity shortly after injury is compara-ble in the three nerve types. In contrast, the postin-jury induction of thrombin-like activity in the nonre-generative optic nerve differs clearly from that inboth regenerative nerves in a way that could explainits detrimental and beneficial effects, respectively, onregeneration.

MATERIALS AND METHODS

Animals

Carp (Cyprinus carpio) were purchased from Tnuva, Israel.Sprague-Dawley rats, 8 wk old, were purchased from theWeizmann Institute of Science Experimental Animal Center.

Animals were used according to the regulations formulatedby IACUC (Institutional Animal Care and Use Committee).

Crush injury

Rats were anesthetized with 10 mg/kg xylazine (Vitamed,Israel) and 50 mg/kg ketamine (Fort Dodge Laboratories,Fort Dodge, Iowa). For the optic nerve crush, a lateralcanthotomy was performed in the right eye under a binocularoperating microscope as described previously (34). The con-junctiva was incised laterally to the cornea, the retractor bulbimuscle was separated, and the optic nerve was exposed. Thedura was left intact. For the sciatic nerve crush, a smallincision was made in the thigh to expose the nerve. Theexposed optic or sciatic nerve was crushed by calibratedforceps for 30 s. After the sciatic nerve crush, the skin wassutured. The rats were allowed to recover.

Fish were deeply anesthetized with 0.05% 3-aminobenzoicacid ethyl ester (Sigma, St. Louis, Mo.). As in the rat, theconjunctiva was incised laterally to the cornea and the ex-posed optic nerve was crushed intraorbitally with forceps for30 s. The fish were then returned to their tanks.

Preparation of conditioned media

At specified times after crush injury, the animals were againanesthetized and their crushed nerves were dissected out,cleaned with Kimwipes, and washed in Dulbecco’s modifiedEagle’s medium (DMEM) without phenol red. Each of thethree types of nerves was pooled and incubated separately for1.5 h at room temperature in DMEM without phenol red. Thenerves were then removed and the three resulting media, nowtermed ‘conditioned media’, were collected, centrifuged at15000 3 g for 15 min in an Eppendorf centrifuge at 4°C, andstored at 270°C. Before use, the conditioned media werekept on ice to limit proteolysis. Protein concentrations weredetermined by Bradford analysis (Bio-Rad, Hercules, Calif.).For some experiments, nerves were separated into proximaland distal segments before incubation. As controls, condi-tioned media were similarly prepared from uninjured nerves.

Preparation of nerve extracts

As in preparation of the conditioned media, the animals werereanesthetized at specified times after crush injury. Thecrushed nerves were dissected from their sheaths and somewere separated, as before, into proximal and distal segments.The pooled nerves or nerve segments were then homoge-nized and left overnight at 4°C in extraction buffer contain-ing 0.5% Triton X-100, 0.1 M Tris-HCl, pH 8.0. After centrif-ugation at 15,000 3 g for 15 min at 4°C, the proteinconcentrations of the supernatants were determined by Brad-ford analysis (Bio-Rad). Nerve extracts were stored at 270°Cand prior to the activity assays were kept on ice to limitproteolysis. As controls, nerve extracts were similarly pre-pared from uninjured nerves.

Thrombin activity assay

An assay for thrombin activity was designed according to apreviously described procedure (35). Samples of the condi-tioned media, each containing 5 mg of total protein, wereincubated at 37°C in a flat-bottomed, 96-well plate with 50mM Tris-HCl, 5 mM EDTA, pH 8.4, and 150 mM of N-p-Tosyl-Gly-Pro-Arg-p-nitroanilidine (Chromozym-TH; Sigma), a spe-cific substrate for thrombin, in DMEM without phenol red ina final volume of 200 ml. In some experiments, the thrombin

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inhibitor hirudin (Sigma) or thromstop (American Diagnos-tica, Greenwich, Conn.) was added. Optical density (OD) at405 nm was measured at the times specified. Wells containingall of the above except for the conditioned media were usedas blanks, and signals in the tested samples were expressed asthe difference between the measured OD and the OD re-corded at the beginning of the assay. Only those measure-ments obtained when enzymatic activity was in the linearrange are shown. In some cases, pure thrombin from bovineplasma (Sigma) was assayed in order to quantify the signals ofthe assay.

Thrombin activation by ecarin

Samples of the conditioned media, each containing 5 mg oftotal protein, were incubated for 2 h at 37°C in a flat-bottomed, 96-well plate with 50 mM Tris-HCl, 5 mM EDTA,pH 8.4, in DMEM without phenol red, in a final volume of100 ml with 0.1 U or 1 U of ecarin (American Diagnostica), aspecific prothrombin activator derived from snake venom.Controls were treated in the same way, but without ecarin.Thrombin-like activity was then assayed as described above.Most of the existing prothrombin was activated by 0.1 U ofecarin, as increasing the concentration to 1 U was found tohave no further effect on the signal.

Gelatin zymography

Conditioned media or nerve extracts containing equalamounts of protein were electrophoresed through an 8%sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel (Bio-Rad) that was polymerized in the pres-ence of 1 mg/ml gelatin. To produce a linear polyacrylamidegradient gel, a 7% and a 15% gel solution—each containing1 mg/ml gelatin—were mixed by a gradient mixer andpoured with a peristaltic pump at a speed of 2 ml/min, asdescribed (36). The gels were washed once for 30 min in2.5% Triton X-100 to remove the SDS, and once for 30 min inthe reaction buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mMCaCl2, pH 7.5). The gels were then incubated in freshreaction buffer at 37°C for 3 days. Gelatinolytic activity wasvisualized by staining of the gels with 0.5% Coomassie bril-liant blue.

Statistics

The significance of differences was calculated using theWelch alternate t test. This test assumes Gaussian populations,but does not assume that the compared populations haveequal standard deviations, and is therefore more conservativethan the conventional unpaired Student’s t test.

RESULTS

Thrombin-like activity in rat optic and sciaticnerves

Thrombin activities were assayed in conditionedmedia prepared from uninjured rat optic and sciaticnerves and from nerves excised 1, 4, and 7 days afterthe injury (Fig. 1A). Samples of conditioned mediawere then incubated for 1 h with the chromogenicsubstrate Chromozym-TH and their OD values wererecorded. Conditioned media derived from optic

Figure 1. Thrombin-like activities in rat optic and sciaticnerves. Nerves were subjected to crush injury, the rats werekilled at the indicated time points after the injury, andconditioned media were collected. A) Thrombin-like activi-ties in rat optic nerve-conditioned medium (ONCM, white)and sciatic nerve-conditioned medium (SNCM, black). Activ-ity-generated elevations in OD at 405 nm after 1 h ofthrombin activity assay are shown. The bar graph shows themean 6sem of OD values obtained from four differentpreparations. The increase in thrombin-like activity on day 1after injury is extremely significant in the optic nerve(***P,0.0001) and very significant in the sciatic nerve(P,0.001) when compared to that obtained in uninjurednerves. Insert: Thrombin-like activity in rat sciatic nerve-conditioned medium. Activity-generated elevations in OD at405 nm after 10 h of thrombin activity assay are shown. Eachbar graph shows the mean 6sem of OD values obtained fromfour different preparations. The increase in thrombin-likeactivity on day 1 after injury is very significant compared tothat obtained in uninjured nerves (**P,0.001). B) Throm-bin-like activitiy in rat optic nerve-conditioned medium(ONCM). Activity-generated elevations in OD at 405 nm after1 h of thrombin activity assay are shown. The graph shows themean 6sem of values obtained from different preparations,whose numbers (n) are indicated at each point. The increasein thrombin-like activity on day 1 after the injury is extremelysignificant (***P,0.0001) compared to that obtained inuninjured nerves.

535GELATINASE AND THROMBIN-LIKE ACTIVITIES IN NERVOUS SYSTEMS

nerves excised 1 day after the injury showed amarked increase in thrombin-like activity relative tothe uninjured optic nerves (P,0.0001) and corre-sponded in intensity to the activity evoked by approx-imately 9 3 1023 U of pure bovine thrombin. Thekinetics are shown in more detail in Fig. 1B. Anincrease in thrombin-like activity levels could bedetected as early as 1.5 h after injury. On day 1postinjury, thrombin levels were about 40-foldhigher than those in control nerves and then grad-ually declined, reaching baseline levels by day 4.

The sciatic nerve also showed an increase inthrombin-like activity 1 day after the injury, but thiselevation was approximately 1/20th that of the opticnerve (Fig. 1A) and could be simulated by 0.5 3 1023

U of pure bovine thrombin. Because these signalswere very low, we extended the incubation time withChromozym-TH from 1 to 10 h (Fig. 1A, insert). Inboth cases, the thrombin-like activity in the sciaticnerve on day 1 postinjury was approximately three-fold higher than in the uninjured sciatic nerve(P,0.001) and had returned to baseline by 4 daysafter injury. Thus, both the sciatic and the opticnerve showed an increase in thrombin-like activity 1day after injury, but the increase in the optic nervewas approximately 20-fold higher than in the sciaticnerve.

Specific inhibition of thrombin-like activity in therat

To further verify the specificity of the measuredthrombin-like activity, we analyzed it in the presence

of the synthetic thrombin inhibitor thromstop (Fig.2) or hirudin (not shown). At the different timepoints examined, similar dose-dependent patterns ofinhibition were observed in the optic and the sciaticnerves: the presence of thromstop at a concentrationof 0.1 mM inhibited activity by approximately 60%,and at a concentration of 1 mM by about 90%.

Thrombin-like activity is potentially associated withretrograde degeneration

To find out whether the elevated thrombin-likeactivity in the injured rat optic nerve on day 1postinjury is associated with retrograde or antero-grade degeneration, some of the nerves were dividedinto a distal segment (between the injury site and theoptic chiasma) and a proximal segment (includingthe site of injury and the part of the nerve betweenthe injury site and the optic disk). Conditionedmedia from the pooled proximal segments and thepooled distal segments were collected separately andtheir thrombin activities were assayed. In all casesexamined, thrombin activity was found mainly in theproximal segments (Fig. 3).

Increase of thrombin-like activity by prothrombinactivation

One possible source for the observed activity couldbe the activation of prothrombin, known to be

Figure 2. Thrombin-like activity in the presence of the specificthrombin inhibitor thromstop in rat optic (gray circles) andsciatic (black squares) nerve. Rats were killed on the day afterinjury and conditioned media were collected. Thrombin-likeactivity was assayed in the presence of different concentra-tions of thromstop. Mean values 6sd of representative exam-ples are shown. Calculation of the inhibition was based on ODelevation at 405 nm after 1 h of thrombin activity assay in theoptic nerve and 10 h of the assay in the sciatic nerve.

Figure 3. Thrombin-like activity in different parts of the ratoptic nerve on day 1 after the injury. Nerves were subjected tocrush injury, the rats were killed 1 day later, and each opticnerve was separated into a proximal segment (which includedthe site of injury) and a distal segment. OD values at 405 nmafter 0.5 h of thrombin activity assay in conditioned media areshown. The bar graph shows the mean 6sem of OD valuesobtained from three different preparations.

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expressed in the nervous system (37). To comparethe amount of prothrombin (a reservoir for throm-bin-like activity) in the rat optic or sciatic nervesbefore and after injury, we measured prothrombinlevels in the various conditioned media. After theaddition of ecarin, which converts prothrombin tothrombin, thrombin-like activity was assayed as be-fore.

In the uninjured optic nerve (Fig. 4A), incubationwith 0.1 U of ecarin did not evoke a thrombin activitysignal. In the injured optic nerve, however, the highsignal observed 1 day after injury was further in-creased approximately threefold by ecarin, suggest-ing that the amount of thrombin-like activity in thenerve after the injury had corresponded to about30% of the potentially available activity. In the sciaticnerve (Fig. 4B), incubation with ecarin increased thesignal of thrombin-like activity by more than 10-foldin both injured and uninjured nerves. Thus, regard-less of injury, it appears that less than 10% of thepotentially available thrombin-like activity in thesciatic nerve exists in the active form.

Thrombin-like activity in fish optic nerve

The kinetics of thrombin-like activity was studied in asimilar manner in the regenerating fish optic nerve.Figure 5A shows the thrombin-like activity measuredin fish optic nerve on different days after injury aswell as in uninjured nerves. Samples of conditionedmedia were incubated with the chromogenic sub-strate Chromozym-TH and the OD values were mea-sured after 4 h. Injury of the fish optic nerve resultedin an increase in thrombin-like activity with kineticsthat differed from those observed in the rat. Theactivity started to increase on day 1, reached a broadpeak that lasted from day 4 to day 7 after the injury,and then declined. The observed increase was signif-icant when compared to that in uninjured nerves(P,0.05).

Specific inhibition of thrombin-like activity in fishoptic nerve

Thrombin-like activity in the fish optic nerve wasinhibited in a dose-dependent manner by the syn-thetic inhibitor thromstop (Fig. 5B) or hirudin (notshown), demonstrating its specificity. Much largeramounts of both inhibitors were needed to achieveinhibition in the fish nerve than in either of the ratnerves: for example, to achieve an inhibition ofabout 60% in the fish nerve it was necessary to use100 mM thromstop, a concentration 1000-fold higherthan that needed in the rat. The inhibition patternin the fish optic nerve was identical at several exam-ined time points.

Activities of gelatinases in rat optic and sciaticnerves

To examine whether the observed up-regulation ofthrombin-like activity in the injured optic nerve ofthe rat is unique to thrombin or merely part of ageneral, nonspecific up-regulation of protease activ-ity after injury (which would imply a general differ-ence in protease regulation between regenerativeand nonregenerative nervous systems), we also mea-sured the postinjury activities of gelatinases. As men-tioned earlier, gelatinases are known to participatein processes that are related to both regeneration(27–29) and degeneration (30–33) in the nervoussystem. Conditioned media and nerve extracts fromrat optic and sciatic nerves were collected at speci-fied times after injury, as well as from uninjurednerves, and gel zymography was performed. Figure 6shows representative gels of gelatinase activities in ratoptic (Fig. 6A) and sciatic (Fig 6B) nerves. In bothcases, gelatinase A (72 kDa) was found to be consti-tutively expressed and did not increase after injury.In contrast, gelatinase B (92 kDa) could not bedetected in either of the nerves before injury, butwas up-regulated in both 1 day after injury, afterwhich its activity declined. In the rat optic nerve, thisup-regulation was detectable as early as 6 h after theinjury (Fig. 6A, lane 2). In some optic nerve prepa-rations, an additional band below 200 kDa was visible(Fig. 7A, lane 1), probably representing a complexformed between a gelatinase and an inhibitor. Apartfrom the existence of this complex, the kinetics ofthe rat sciatic and optic nerves were similar. Identicalresults were obtained when nerve extracts were usedinstead of conditioned media (not shown).

Figure 4. Prothrombin levels and thrombin-like activities inrat optic (A) and sciatic (B) nerves. Nerves were subjected tocrush injury, the rats were killed 1 day later, and conditionedmedia were collected from injured and uninjured nerves. Forconversion of prothrombin to thrombin, 0.1 U ecarin wasadded prior to the thrombin activity assay. Representativeexamples (mean 6sd) of OD values at 405 nm after 0.5 h ofthe assay are shown.

537GELATINASE AND THROMBIN-LIKE ACTIVITIES IN NERVOUS SYSTEMS

Gelatinase activity is potentially associated withretrograde degeneration

Having found that thrombin-like activity in the ratoptic nerve is potentially associated with retrograde

degeneration (Fig. 3), we were interested in estab-lishing which parts of the injured optic and sciaticnerves are responsible for the observed changes ingelatinase activity. Accordingly, both nerves wereagain divided into a proximal segment, which in-cluded the site of injury, and a distal segment. Nerveextracts were prepared from each segment sepa-rately. In both optic and sciatic nerves, induction ofgelatinase B was found to occur only in the proximalsegment (Fig. 7). Identical results were obtainedwhen conditioned media were used (not shown).

Gelatinase activities in the fish optic nerve

As with thrombin, we studied the kinetics of gelati-nase activities in response to axonal injury in theregenerating fish optic nerve. As before, conditionedmedia and nerve extracts were collected at specifiedtimes after the injury, as well as from uninjurednerves, and gel zymography was performed. Figure 8shows representative gels of gelatinase activities inthe fish optic nerve. Two major bands were found at72 and 92 kDa. Since they were identical in size andexhibited similar postinjury behavior to that of thegelatinase activity observed in the rat, they wereassumed to be homologous to rat gelatinases A andB, respectively. As in the rat, the 72 kDa gelatinasewas found to be constitutively expressed and unaf-fected by the injury, whereas the 92 kDa gelatinasewas up-regulated. Several additional phenomena,not present in the rat, were observed in the fish. 1)Up-regulation of gelatinase activity after injury re-mained high for much longer than in the rat; forexample, baseline levels of the 92 kDa gelatinasewere not reached even by day 7. 2) In uninjurednerves, the ratio between the gelatinases at 72 and 92kDa differed from that obtained in the rat, indicat-

Figure 5. A) Thrombin-like activity infish optic nerve. Nerves were sub-jected to crush injury, the fish werekilled on the indicated days afterinjury, and conditioned media werecollected. Activity-generated eleva-tions in OD at 405 nm after 4 h ofthrombin activity assay are shown.The bar graph shows the mean val-ues 6sem of OD values obtainedfrom different preparations, whosenumbers (n) are indicated aboveeach bar. The increase in thrombin-like activity on days 4 and 7 after theinjury is significant (*P,0.05) com-pared to that obtained in uninjurednerves. B) Thrombin-like activity inthe fish optic nerve in the presenceof the specific thrombin inhibitorthromstop. Fish were killed on day 7 after injury, conditioned medium was collected, and thrombin activity assay wasperformed in the presence of different amounts of thromstop. Mean OD values 6sd of a representative example areshown. Calculation of the inhibition was based on OD elevation at 405 nm after 4 h of thrombin activity assay.

Figure 6. Gelatinase activities in rat optic (A) and sciatic (B)nerve. Nerves were subjected to crush injury, the rats werekilled at the indicated times after the injury, and conditionedmedia were collected. Gel zymography in 8% gels was per-formed with 10 mg of total protein. MMP-2, gelatinase A;MMP-9, gelatinase B.

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ing a greater abundance of the 92 kDa gelatinase inthe fish. 3) Additional bands at about 20, 35, 45, 55,and 80 kDa were up-regulated upon injury. Thesebands might be a result of up-regulation of addi-tional gelatinases or of proteolysis of the knowngelatinases where the cleaved products were stillactive. 4) Less protein was needed in the fish condi-tioned medium in order to achieve a band intensitysimilar to that of the rat conditioned medium,indicating that the activity in the former medium ishigher, either because gelatinases are more abun-dant or because the activities of the fish gelatinasesare higher than those of the rat. Identical resultswere obtained when nerve extracts were used insteadof conditioned media (not shown).

DISCUSSION

The neuronal response to injury, as manifested bytwo types of proteolytic activities, was compared inthe nonregenerative rat optic nerve and two regen-erative nerves: the rat sciatic nerve and the fish opticnerve. In the rat optic nerve, an extremely significantincrease in thrombin-like activity (approximately 40-fold compared to that in the uninjured nerve) wasobserved 1 day after injury. The increase was tran-sient and started to decline 2 days after injury,returning to baseline levels on about day 4. In the ratsciatic nerve, a transient threefold increase in throm-bin-like activity was seen 1 day after injury. Althoughthis increase was about 20-fold smaller than in theoptic nerve, it was still very significant. In both theoptic and the sciatic nerves of rat, the thrombin-likeactivity was inhibited in a dose-dependent manner bytwo specific inhibitors, thromstop and hirudin, con-firming the specificity of the observed signal.

High levels of thrombin activity were previouslyshown to inhibit neurite outgrowth (16) and toinduce apoptotic cell death in neurons and astro-cytes (20–22) in vitro. The high levels of thrombin-like activity in the rat optic nerve observed 1 day afterinjury in our study suggest that the degenerativeprocesses seen after CNS injury might be related tothrombin activity and that thrombin-like activitymight contribute to the nonregenerative nature ofthe optic nerve environment. The marked increase

Figure 7. Gelatinase activities in different parts of rat optic (A)and sciatic (B) nerve. Nerves were subjected to crush injury,the rats were killed on the indicated days after injury, andnerve extracts were collected. Gel zymography in 8% gels wasperformed with 30 mg of total protein. P, proximal segment;D, distal segment; MMP-2, gelatinase A; MMP-9, gelatinase B.

Figure 8. Gelatinase activities in fish optic nerve. Nerves weresubjected to crush injury, the fish were killed on the indicateddays after injury, and conditioned media were collected. A)Gel zymography in a 7–15% linear gradient gel was per-formed with 10 mg of total protein. B) Gel zymography in 8%gel was performed with 2.5 mg of total protein.

539GELATINASE AND THROMBIN-LIKE ACTIVITIES IN NERVOUS SYSTEMS

in activity could also override the beneficial effects ofthrombin that are expected (from in vitro data) tooccur in the CNS, such as the stimulation of NGFsecretion by astrocytes (12). In contrast, a slightlyincreased thrombin-like activity, such as that seen inthe rat sciatic nerve, might not only be below thetoxicity threshold but might even be beneficial forthe injured nerves. In the PNS, the injured nerve israpidly invaded by macrophages, which are crucialfor regeneration in the nervous system (1, 38–40).Thrombin is a potent chemoattractant for humanmonocytes (41) and stimulates the production ofmonocyte chemotactic proteins (42–44). In themammalian CNS, where macrophage recruitment isimpaired (38) because of an immune brain barrier(45), these potential effects of thrombin could beminimized or excluded. In addition, levels of throm-bin-like activity in the sciatic nerve could be withinthe range at which neurons are rescued from celldeath resulting from metabolic insults, which likelyto be present after nerve injury (20, 21).

Although it cannot be ruled out, the possibilitythat the observed thrombin-like activity results froma protein other than thrombin seems very unlikely,an assumption supported both by the cleavage of thespecific thrombin substrate Chromozym-TH and theinhibition by two specific thrombin inhibitors.Thrombin activity results from the cleavage of pro-thrombin, which is known to be expressed in thenervous system (37). The experiments with ecarinshowed that activation of the available prothrombinwould increase thrombin activity by more than 10-fold in the injured sciatic nerve, but by only about3-fold in the injured optic nerve. This suggests thatprothrombin activation is under tighter control inthe sciatic nerve than in the optic nerve. A prothrom-bin activator independent of the blood coagulationcascade was recently described, and its involvementafter injury was suggested (46, 47). Although notdetected in uninjured brain, it was found to be activein a cell line derived from mammalian CNS (46). Inaddition, prothrombin activation has been shown tooccur on neuronal surfaces (48). Our data suggestthat different mechanisms of prothrombin activationin the optic and sciatic nerves could account for thehigh levels of thrombin-like activity observed afterinjury in the rat optic nerve.

In a recent study of thrombin and prothrombinelevation in rat sciatic nerve (49), thrombin-gener-ated activity increased significantly from day 1 afternerve crush, reaching a peak on day 3 and returningto basal levels on day 6. Prothrombin could not bedetected in uninjured nerves, but the ratio of pro-thrombin-to-thrombin activity after injury was com-parable to the ratio obtained in our study, and ledthose authors to suggest a tight control of thrombinactivation in the sciatic nerve. The different kinetics

obtained in that study (reflecting a relatively delayedincrease in thrombin activity) and the inability todetect prothrombin in the uninjured sciatic nervemight be attributable to the methodology, as nerveextracts and not conditioned media were used.

Differences in thrombin-like activities between therat optic and sciatic nerves could also result fromchanges in the levels of thrombin inhibitors. Aknown thrombin inhibitor in the nervous system isthe glial-derived protease nexin I (PN-I) (50), whichpromotes neurite outgrowth (51, 52) and couldtherefore reverse the negative effects of thrombin-like activity. In the sciatic nerve, PN-I is up-regulatedafter injury, starting on about day 3 and peaking onday 7 (49, 53). The behavior of PN-I shortly afterinjury in the optic nerve has not been studied.Nevertheless, our finding of high thrombin-like ac-tivity in the optic nerve 1 day after injury may reflectthe paucity or absence of thrombin inhibitors, suchas PN-I, at this time.

In the fish, the kinetics of thrombin-like activity inthe optic nerve differed from those in the rat. As inthe rat, the increase in activity started on day 1 but,unlike in the rat, continued and reached a plateaulasting from day 4 to day 7 after injury. As similarpeaking in the fish nerve was also observed in theactivities of the gelatinases, this elevation patternmay represent a general response of this regenera-tive nerve to injury. The kinetics observed in the fishoptic nerve point to the establishment of a differentextracellular milieu, which is generated by the pro-teases and could account, at least in part, for theregeneration-supportive properties of the neuronalenvironment. As in the rat, thrombin-like activity inthe fish optic nerve was specifically inhibited, in adose-dependent manner, by the inhibitors throm-stop and hirudin. Much larger amounts of theseinhibitors were needed in the fish than in the rat,indicating the presence of other substances in thefish conditioned medium that influence the interac-tion between the inhibitors and the enzyme.

Studies in our laboratory recently demonstratedthe presence of factor XIIIa in the nervous tissues ofrat and fish, as well as a correlation between thepostinjury appearance and activation of the enzymeand the regenerative ability of the tissue (54).Thrombin is crucial for the regulation of factor XIIIain vivo, and the differential activities of thrombinmight be responsible for the differences in theobserved behavior of factor XIIIa.

We found that the thrombin-like activity in the ratoptic nerve was associated mainly with the part of thenerve adjacent to and including the site of injury,i.e., the proximal segment. This suggests that theobserved injury-induced activity is associated withretrograde degeneration and, accordingly, is evokedeither by changes emanating from the neuronal cell

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bodies or from a non-neuronal cell response at thesite of injury. As mentioned earlier, prothrombinmRNA was shown to be expressed by cells of thenervous system (37). In addition, thrombin has beendetected in brain and astroglial cell cultures (55).Nevertheless, thrombin synthesis could also occurelsewhere, for example, in platelets that adhere tothe site of injury. The cellular source responsible forthrombin activity in the nervous system, and partic-ularly after injury, is not yet known.

In contrast to the differences in thrombin-likeactivity, we found that the activation pattern ofgelatinase A or B was similar in all three nerve types.Gelatinase B was up-regulated after crush injury,whereas gelatinase A was not affected. In the rat,up-regulation of gelatinase B (which did not occur inthe uninjured nerve) peaked about 1 day after opticor sciatic nerve injury and decreased on about day 4.The fish showed a different proportion between thetwo gelatinases in the uninjured nerve, as well as arelatively delayed peak of the activity. As alreadymentioned, this delayed peaking (which is not exclu-sive to gelatinases) may influence the regenerativeproperties of the fish optic nerve.

Gelatinases are known to participate in processesthat are related to both regeneration (27–29) anddegeneration (30–33) in the nervous system. Thisstudy shows not only that gelatinases are present inregenerative and nonregenerative white matter butthat, surprisingly, the induction patterns of gelati-nases A and B in all three nerve types examined aresimilar. One possible explanation is that rather thanbeing directly related to degeneration or regenera-tion, the mechanisms of activation or inhibition ofgelatinases in degenerating and regenerating sys-tems might differ. Gelatinase activity can be inhib-ited by tissue inhibitors of matrix metalloproteases(TIMPs). TIMP-1 was shown to be induced in thesciatic nerve on days 1 and 4 postinjury, and immu-nostaining demonstrated its colocalization withSchwann cells and macrophages (56). Another pos-sibility is that not only total protease activity, but alsothe ratio of ECM-degrading to ECM-producing pro-teases, might play a role in determining the permis-siveness of the extracellular milieu to regeneration.This is in line with our finding that the nonregen-erative rat optic nerve differed significantly from theregenerative rat sciatic nerve with respect to theamounts of fibrin-producing thrombin, but not ofECM-degrading gelatinases.

As with thrombin-like activity, gelatinase activitiesin the rat optic and sciatic nerve were limited to theproximal segment, indicating that the observed ac-tivities are associated not with anterograde degener-ation, but with an active process occurring at the siteof injury or in the proximal part of the nerve. Ourfindings in the sciatic nerve are in line with the

observations of La Fleur et al. (56). The cellularsource of the gelatinases in vivo remains to beestablished. However, the inflammation-related cyto-kines interleukin (IL) -1a, IL-1b, and TNF-a arepotent inducers of gelatinases in cultured rat astro-cytes (57), and activated microglia release gelatinaseB in vitro (58). Gelatinase activity has also beenreported in Schwann cells and glioma cells (59, 60).

In summary, a comparison of the general kineticsof the induced thrombin-like and gelatinase activi-ties in rat and fish showed that activity in the ratpeaks early and declines between days 1 and 4postinjury, whereas in the fish the activity lasts for atleast a week. A comparison of activity levels in theoptic and sciatic nerves of the rat showed significantdifferences in thrombin-like activity but similarity inthe activities of gelatinases. The induction pattern ofthe 72 kDa and 92 kDa gelatinases was similar in fishas well. Both the different kinetics and the differen-tial activity levels might affect the nerve’s ability toregenerate. This study further highlights the factthat tissue repair and regeneration are closely linkedand that substances that generally participate inwound healing establish an extracellular milieu thatcan lead either to regeneration or its failure.

We thank Shirley Smith for editorial assistance and HayaAvital for help with graphics. M.S. holds the Maurice and IlseKatz Professorial Chair in Neurobiology.

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Received for publication July 27, 1998.Revised for publication October 29, 1998.

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