(C) Freund Publishing House Ltd., 1995

Fibrin Glue Used as an Adhesive Agent in CNS Tissues

Henrich Cheng’2, Susanne Almstr6m and Lars Olson

Department ofNeuroscience, Karofinska Institute, Stockholm, Sweden2Department ofNeurosurgery, Neurologic Institute, Veteran General Hospital-Taipei

and Division ofSurgery, National Yang-Ming Medical College, Taiwan, R. O. C.

SUMMARY

One of the limitations of many bridgingexperiments in neural transplantation is that theCNS tissues cannot be sutured. Fibrin glue is atwo-component system derived from wholeblood which, when mixed, reproduces the finalstage of blood coagulation and solidifies. Manyexperimental studies of humans and animalsshow that fibrin glue repair of peripheral nervesis almost equivalent to microsurgical sutures. Inthis study, we attempted to extend its use toCNS tissues and transplants. Two techniqueswere tried: (1) Bilateral parietal knife cuts wereperformed by stereotaxic technique in six rats.Fibrin glue was applied in the right-side corticallesion. Immunohistochemistry using antisera totyrosine hydroxylase (TH), glial fibrillary acidicprotein (GFAP), laminin and neurof’dament(NF) was essentially similar between the controland treatment groups. The immunoreactivity ofeach marker revealed no significant differencesbetween the two groups on days 1, 7 and 30.There was no difference in terms of gliosis ormicrovascular proliferation. (2) Embryonic day16 fetal locus coeruleus was grafted togetherwith El6 cortex to the anterior chamber of sym-pathectomized eyes. In the six eyes of the gluetreatment group, the parietal cortical piece andthe locus coeruleus piece were joined togetherbefore grafting by immersing them in thesolution of fibrin glue. In the eight eyes of thecontrol group, pieces of parietal cortex and locuscoeruleus were introduced individually and ap-proximated by gently pressing the cornea. The

Reprint address:Henrich Cheng, M.D.Histology, Department of NeuroscienceKarolinska InstituteS- 171 77 Stockholm, Sweden

sizes of double grafts showed no significantdifference between groups during six weekspostgrafting. The immunohistochemical picturesusing antisera against TH, GFAP and lamininwere similar in both groups. Catecholaminergicfibers from the grafted locus coeruleus werefound bridging over into the parietal corticalpiece in both the control and treatment groups.There was no significant difference in TH-positive nerve fiber density between tissue glue-joined and control double intraocular grafts. Inconclusion, fibrin glue can be used as an ad-hesive agent in CNS tissues without hamperingthe outgrowth of neurites or causing adversetissue reactions in fetal or adult nervous tissues.

KEY WORDS

fibrin glue, CNS repair, brain development,transplantation, locus coeruleus, cortex cerebri

INTRODUCTION

In the intensive search for methods to improvethe regenerative capability of CNS tissues, manydifferent bridging models have been attempted. Oneof the limitations of such experiments is that CNStissues cannot be sutured. The CNS microvascu-lature cannot sustain suturing and the fragility ofthetissue makes attempts to use stitches hazardous oruseless. One approach uses transplants which mayserve as bridges for regenerating axons across a siteof injury/25/. Alternative methods for bridging byinserting peripheral nerve pieces, or lodging tissuesinto the CNS, or anchoring to the dura matter withmicrosutures could provide moderate but not strongjoining.

For peripheral nerve repair, several suturelessmethods have been developed, including fibrin glue

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and laser coaptations /6,8,10-12,14,22,26/. Fibringlue, or tissue glue, is a two-component systemderived from whole blood which, when mixed,reproduces the final stage of blood coagulation toform a viscous adhesive. This glue is commerciallyavailable and used both clinically and experimentallyin Europe and Japan. It consists of highly concen-trated human fibrinogen which, after adding apro-tinin, reacts with the calcium-activated thrombin toform a sealant that can maintain tissue approxi-mation. Most ofthe experimental studies on humansor animals have shown that fibrin glue repair ofperipheral nerves was equivalent to microsurgicalsutures/15,23,27/. Its major advantage over suturesis that the time required for the procedure is shorterand the anastomosis is easier to perform, especiallyin hard-to-access areas.

In this study, we attempted to extend the use offibrin glue to CNS tissues and transplants. First, arodent CNS lesion model was used to evaluate anyadverse effect of the glue in lesioned CNS areas.Next, we used the fibrin glue as an adhesive agentbetween two pieces ofintraocular fetal grafts (locuscoeruleus and cerebral cortex) to evaluate itsinfluence on survival and growth of fetal tissuegrafts. Finally, the intraocular double graft modelallowed us to determine whether nerve fibers couldbridge a layer of fibrin glue at the interface betweentwo brain tissue areas.

MATERIALS AND METHODS

Fibrin glue

The fibrin glue used for the present study was afibrinogen-based compound with double sealantcomponents (Beriplast(R)P, Behring, BehringwerkeAG, Marburg, Germany). The 1 ml Beriplast(R)P setcontains:Vial 1" Fibrinogen concentrate consisting of 115-232 mg dry substance, containing a human plasmaprotein fraction with 65-115 mg fibrinogen and ahuman plasma protein fraction with a factor XIIIactivity of40-80 U.Vial 2- Aprotinin solution consisting of 1 ml sol-ution containing 1000 KIU ofbovine lung aprotinin.Vial 3" Thrombin consisting of 4.9-11.1 mg dry

substance containing a human plasma proteinfraction with a thrombin activity of400-600 IU.Vial 4" Calcium chloride solution consisting of 2.5ml solution containing 14.7 mg calciumchloride.2H20 (40 mmol/1).

Before application, the two sealant elementswere prepared separately by transferring the apro-tinin solution to the fibrinogen concentrate andtransferring the calcium chloride solution to thethrombin. To apply the fibrin glue, a tuberculinsyringe was filled with each sealant element. Bypushing the two syringe plungers simultaneously, acoagulating glue forms between the tips.

Fibrin glue in a CNS lesion model

Adult 150-g female rats (Sprague-Dawley, B&KUniversal, Stockholm) were used to study the effectof fibrin glue in the CNS lesion model. Undergeneral halothane anesthesia, the animal wasmounted in a stereotaxic frame. A sharp blade witha flat cutting edge, 2 mm in length, was clampedonto the frame. Bilateral parietal craniectomies wereperformed to expose 3 x 3 mm2 areas on both sides.The central bony ridge over the sagittal sinus waspreserved to impede the liquid flow from one sideto the other. Under stereotaxic guidance, thecenters ofthe craniectomies were identified for cor-tical incision (AP 1.5, L/R 1.5, V 0 from bregma).The dura was opened and bilateral cortical incisionswere performed by moving the clamped blade 3 mmdownward (from V 0 to V 3, see Fig. 1). Thecontonting vessels on the cortical surface werecauterized and severed before cutting to preventprofuse bleeding. Fibrin glue was applied into theright-side cortical lesion. Care was taken to avoidany fluid leak to the left side. After the glue hard-ened, the craniectomies were covered by pieces ofspongostan and the wound closed.

The six rats were grouped into three groups andsacrificed on days 1, 7 and 28 respectively. Theanimals were anesthetized with sodium pentobar-bital (Mebumal 40 mg/kg i.p.) and perfused via theascending aorta with 50 ml calcium-free Tyrode’ssolution followed by 50 ml formalin-picric acidmixture (4% paraformaldehyde, 0.4% picric acid in0.16 M phosphate buffer, pH 7.4). Brains wereremoved and processed for indirect immunohisto-chemistry using antibodies against tyrosine hydro-

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FIBRIN GLUE 235

Fig. 1:

AI}rottnm Fil)rmogenSotuton Concentrate

CC

ThromOin CalciumChlorideSolution

Schematic diagram illustrating; the procedures for double intraocular grafts. Pieces of parietal cortex (CC) and locuscoeruleus (LC) were dissected out from the El6 fetal brain. In the treatment group, a cortical piece and a locuscoeruleus piece were joined together by immersing them in the unhardened solution of fibrin glue. The glue-joineddouble grafts were introduced into the anterior chambers of sympathectomized eyes. Six weeks later, the grafts wereremoved for immunohistochemical examination. C: parietal cortical piece, L: locus coeruleus piece.

xylase (TH), glial fibrillary acidic protein (GFAP),laminin, and neurofilament protein (NF). Tissueswere examined and photographed using epifluores-cence microscopy (Nikon-Microphot). Densities ofTH, GFAP, larninin and NF imrntmoreactivitieswere semiquantitatively scored on all sections.Averages from 6-10 sections from a given brainwere then calculated. A semiquantitative scale from0-5 was used.

Fibrin glue in double intraocular gratis (locuscoeruleus + cortex cerebri)

Young adult 150-g female rats (Sprague-Dawley, B&K Universal, Stockholm) were used ashosts to study the effect of fibrin glue in doubleintraocular grafts. All eyes were sympatheticallydenervated by superior cervical ganglionectomy oneweek before transplantation. Fetuses from pregnant

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FIBRIN GLIJE 237

Immunohistochemistry of lesioned cerebral cortices on days 1, 7 and 30. The histologic pictures with four differentimmunoreactive markers were similar between the control (right side) and the glue-treated (left side) cortices. GFAP:glial fibrillary acidic protein, LAM: laminin, NF: neurofilament protein, TH: tyrosine hydroxylase, a: day 1;b,d and f:day 7;c,e and g: day 30. Scale bar 500 tm in LAM. Scale bar-- 100 gm in GFAP and NF.

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rats of the same strain were used as donors of CNStissue grafts. Pieces of parietal cortex and locuscoeruleus from E16 fetal brains were dissected out.In the treatment group, the cortical and locuscoeruleus pieces were joined together by immersingthem in the fibrin glue solution. The superfluousglue around the joined piece was trimmed after theglue had hardened. After pretreatment of all eyeswith a drop of 1% atropine solution, intraoculargrafting was performed under ether anesthesia.Grafts were introduced through a tiny opening ofthe cornea as previously described/19-21/using amodified Pasteur pipette. The fibrin glue-treatedcombined graft was introduced as a single piece,while in the control group pieces of parietal cortexand locus coemleus were introduced individuallyand juxtaposed by gentle pressure on the cornea.After grafting, the volumes of the transplants weremeasured at regular intervals by stereomicroscopy.The graft size was estimated by taking the longestdiameter of the graft multiplied by the diameterperpendicular to it /4/. Eleven animals were usedwhich consisted of six eyes in the treated group,eight eyes in the untreated group and six eyes ofcortico-cortical glue-treated double grafts asnegative controls (see Fig. 1).

The animals were perfused six weeks aftergrafting using procedures as stated above andprocessed with the same methods for indirect im-munohistochemistry using antibodies against TH,GFAP and laminin. Scoring of TH-immunoreactivenerve fiber density, GFAP-like, and laminin im-munoreactivities was performed on all sections.Averages from 6-10 sections from a given graftwere then calculated to generate means of TH-immunoreactive nerve fiber density, GFAP-like andlaminin immunoreactivity per section. A semiquanti-tative scale from 0-5 was used.

Statistical analysis

Fibrin glue in CNS lesion model

The data were analyzed by nonparametric Mann-Whitney tests. Within each group, the data from theimmunoreactivity of glue-applied sides were com-pared with the data from the correspondingimmunoreactivity of control sides.

Fibrin glue in double intraocular grafts (locuscoeruleus + cortex cerebri)

Graft sizes were analyzed by single-factorrepeated measures ANOVA test. The univariateanalysis was based on repeated measuremems onthe same rats. Number of days after surgery andnumber of eyes were treated as within-subjectfactors. Rats were grouped within the treatmentgroups. The type of treatment was a between-subject factor. The measurements of TH-immuno-reactive nerve fiber density, GFAP-like and lamininimmunoreactivity were analyzed by ANOVA.

RESULTS

Fibrin glue in CNS lesion model

The fight parietal cortex cuts which receivedfibrin glue were compared with the left parietalcortices that received lesions only. The patterns ofimmunohistochemistry using antisera against TH,GFAP, laminin and NF were similar between thecontrol and treatment groups on days 1, 7 and 30(Fig. 2). There was no difference in the degree ofgliosis or microvascular proliferation. No morpho-logic evidence of any special tissue reaction couldbe found in the treatment group. Using the semi-quantitative data, none of the immunoreactivemarkers revealed in the glue group differed signifi-cantly from the controls on days 1, 7 or 30 (Fig. 3).

Fibrin glue in double intraocular gratis (locuscoeruleus + cortex cerebri)

Growth ofdouble intraocular grafts

With or without fibrin glue, joined fetal (El6)locus coemleus and cortex cerebri double pieceswere grafted to the anterior chamber of the eye ofsympathetically denervated host rats. All but two ofthe gratis (in the group of cortico-cortical gratis)survived well and became vascularized from thehost iris. As shown in Fig. 4, there was no signifi-cant difference in volume change between the glue-treated and untreated locus coeruleus plus cortexcerebri gratis (P 0.25 on day 5, P 0.38 on day14, and P- 0.52 on day 25, ANOVA).

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FIBRIN GLUE 239

IR-density 2

day

IR-density

week

2,5

IR-density

month

LAM GFAP NF THr- Control1 Glue-treated

Fig. 3: Comparison of the immunoreactivity between glue-treated and control lesioned cortex with four im-munohistochemical markers based on 6-10 serialsections from each brain (n 2 for each group).The density of neural, glial and vascular elementswith four immunohistochemical stains was similarin the glue-treated and control lesioned cortex ondays 1, 7 and 30. The fiber density of each sectionwas graded from 0 to 5. LAM: laminin, GFAP:glial fibrillary acidic protein, NF: neurofilamentprotein, TH: tyrosine hydroxylase.

EE 5

-= 4

" 3

d

1 ------- C

0 10 2 30

days postgrafting

Fig. 4" Volume of double intraocular grafts (locuscoeruleus + cortex cerebri, E16) on days 5, 14 and25. No significant difference in volume change wasnoted between the glue-treated and untreated locuscoeruleus plus cortex cerebri grafts (glue-treateddouble grafts, n=6; untreated controls, n=8;ANOVA).

Effects of fibrin glue on TH-positive nerve fiberdensity, GFAP and laminin immunoreactivity inintraocular double grafts

The catecholaminergic fibers in the intraoculardouble grafts were examined using TH immuno-histochemistry. There was no significant differencein TH-positive nerve fiber density between glue-joined and untreated locus coeruleus plus cortexcerebri grafts (Figs. 5, 6; P 0.76, ANOVA).Comparing the TH-positive fiber density in theabove glue-treated or untreated locus coeruleusplus cortex cerebri grafts with the glue-treatedcortico-cortical grafts, significant differences werenoted (P 0.017 for the untreated and cortico-cortical grafts, P-- 0.01 for the glue treated andcortico-cortical gratis, ANOVA; Fig. 5). Studies ofGFAP and laminin immunoreactivity in the graftsrevealed no obvious differences between thedifferent treatments (see also Fig. 5).

DISCUSSION

The fact that no morphologic difference could befotmd between the fibrin glue-treated and controllesioned cortex suggests that fibrin glue does notproduce harmful effects in CNS tissue. Gliosis and

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240 H. CtIENG ET AL.

Fig. 5: Immunohistochemistry of double intraocular grafts 37 days postgrafting. The TH-positive nerve fiber density wassimilar between glue-joined (upper) and untreated (lower) locus coeruleus plus cortex cerebri grafts. Scale bar 200tm.

microvascular proliferation did not increase in theglue-treated lesioned cortices in the early, inter-mediate or late periods after injury. Studies of THand NF immunoreactivity also indicated that neuralcomponents of the CNS tissue are unaltered afterthe application of glue. As shown by many authors/1-3,8-11,13,14,16,22,24,26,28/, fibrin glue pro-duces no adverse tissue reaction in PNS tissues.After axotomy, axons can penetrate the glue regionand grow in the distal stumps.

The major controversy concerning the appli-cation of fibrin glue in peripheral nerve repair iswhether it can replace the conventional microsuturetechnique. Studies by several authors/3,9,10,14,22/have shown that the effect of fibrin glue in nervecoaptation is comparable to microsutures in tensilestrength, regenerative axonal counts and, althoughslower in conduction velocity, electrophysiologicresponses. The advantages of fibrin glue in surgeryare rapidity of application, good hemostasis, easier

stabilization of small gratis and possibility of ana-stomosis in some difficult anatomical positions/8,15,22/. The disadvantage ofpossible bacteria andvirus transmission has been resolved by inactivation.Screening for hepatitis B antibodies since the early1980s and for AIDS virus antibodies since the1990s has been done to scrutinize both sourcematerial and the final product/15/. With a relativelyaseptic technique, as presented in this study, infec-tion is not a problem with the use of fibrin glue inthe CNS tissues.

The vulnerable nature ofthe microvasculature ofCNS tissue, the fragile texture of the parenchymaand the sophisticated neural networks combine tomake suturing impossible for reparative inter-vention, qae present htraocular double grafting ex-periments have demonstrated a possible role forfibrin glue to act as a CNS adhesive agent to glueCNS tissues together. The glue-joined fetal doublegrafts survived and grew on the iris. Statistical

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FIBRIN GLUE 241

IR-density

3,5

2,5

1,5

T

TH+fibe GFAP Lam

Comparison of TH-positive nerve fiber density,GFAP-like and laminin immunoreactivity betweenglue-joined double intraocular grafts (G: locuscoeruleus + cortex cerebri, E16, n=6) and untreateddouble intraocular grafts (C: locus coeruleus +cortex cerebri in HBSS, El6, n=8). Double piecesof cortex cerebri (Co + Co: cortex cerebri + cortexcerebri, E16, n=4), joined by fibrin glue, were alsoused as negative controls. There is no significantdifference in TH-positive nerve fiber densitybetween tissue glue-joined and untreated doubleintraocular grafts. Significant differences are notedbetween the above two groups and the cortico-cortical grafts (P=0.0165 for C; Co + Co, P=0.0122for G; Co + Co, ANOVA). No difference is seen inthe density of GFAP-Iike and laminin immuno-reactivity within the three groups (ANOVA).

analysis showed no significant difference betweenthe two groups regarding graft volumes. The studyof TH-positive nerve fiber densities of the intra-ocular double gratis six weeks after graftingrevealed no significant difference between the glue-treated and the untreated groups. Likewise, GFAPand laminin immunoreactivity also appeared similar.Since TH-positive fibers found in the locuscoeruleus plus cortex cerebri gratis could not bederived from the sympathectomized iris, theirpresence in the cortex part of the double graftproves that a coeruleo-cortical TH-positive pathwayhad formed. Clearly, the fibrin glue did not interferewith the growth ofnerve fibers; nor did it cause anyuntoward gliosis or disturbed microcirculation inthe gratis.

Study of the ultrastmcture of fibrin glue withconfocal laser 3D microscopy, li.quid permeationand turbidity/5/shows that the glue from normalfibrinogen is composed of straight rod-like fiberelements which sometimes originate from densernodes. The tautness and porosity of the glue net-works can be altered by changing the concentrationof thrombin and fibrinogen. Thus increasing eitherthrombin or fibrinogen concentrations will cause theglue networks to become tighter and the porosity tobe decreased. The fiber strands also become shorter.Glue porosity of the network can also be modifiedby changing the ionic strengths, coexisting proteins(e.g. albumin, lipoprotein), dextran, etc.

For the catecholaminergic cells in the locuscoeruleus graft to survive and grow, nutrients anddiffusible factors (such as trophic factors, fromeither the iris or the co-grafted cortical piece) mustbe able to reach the cells in the graft/18-21/. Theendothelial cells from the iris must also have accessto the graft, otherwise the graft would degenerateand die. The present study supports the finding thatnutrients and trophic factors can pass through thefibrin glue networks to reach the graft. It means,with the present formula, the formed fibrin polymerscreate a mesh that will hold two graft piecestogether but also permit the transmission ofessential molecules through its pores. The endo-thelial cells from the iris can penetrate the fibrinmeshwork around the graft and finally establish themicrovasculature. Likewise, TH-positive fibers fromthe locus coeruleus can also penetrate the fibrin gluein between the two grafts and reach the corticalpiece. The fibrin meshwork is known to be absorbedby the fibrinolytic system (e.g. plasmin) one weekaer application. The feasibility for the lysis offibringlue can also be altered by structural modificationof the extended dimeric multidomained fibrinmolecule (e.g. as shown in the studies of Blombncket al. and Nossel & Kaplan/5,17/, a scheme invol-ving fibrin I and fibrin II, in which the former is theresult of releasing the fibrinopeptide only. Fibrin IIlacks both fibrinopeptides A and B and is thought tobe involved in lateral anchoring; as a consequence itis more resistant to fibrinolysis), presence ofphysio-logical stabiling factor XIIIa, interactions betweenplasminogen, thrombin and fibrin, cross-linkedfibronectin in the tissues, platelets, etc./5,7,17,25/.

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In the anterior chamber ofthe eye, the fibrin glueis exposed to the chamber fluid, the iris, locuscoeruleus and cortical grail. Since there was nosignificant difference in graft growth or final sizesbetween glue-treated and control double grafts, itappears as if the glue coat did not significantlyimpair the rate of formation or amount of vascularconnections between host iris and gratis. The lysisofthe glue will be affected by numerous factors andinteractions. The timing of the penetration of endo-thelial cells and neurites, in relation to fibrinolysis,remains to be further analyzed.

ACKNOWLEDGEMENTS

The BeriplastP fibrin glue was kindly suppliedby Behringwerke AG, Germany. The study wassupported by the Swedish Medical ResearchCouncil 03185, USPHS grants NS09199 andAG04418, Taiwan Chin-Lin Medical Research Fundand NSC77-0412-B075-17.

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