Role of tissue typing on preserved nerve allografts in...

Journal of Neurology, Neurosurgery, and Psychiatry, 1977, 40, 865-871

Role of tissue typing on preserved nerve allograftsin dogsR. SINGH, K. MECHELSE, AND S. STEFANKO

From the Departments of Neurosurgery, Electroneurology, and Neuropathology, AcademischZiekenhuis Rotterdam-Dijkzigt, Erasmus University, Rotterdam, the Netherlands

S U MM AR Y Histocompatibility seems to play an important role in the regeneration of nerveallografts. Compatible tissue typed nerve allografts behave more like autografts and are, there-fore, more readily accepted by the host tissue without producing evident tissue rejection. Theinfluence of histocompatibility difference becomes more marked if the graft is longer than30-40 mm. Irradiation as a means of reducing the immune reaction of the nerve allograftsdqes not seem to have any beneficial effect along with tissue typing. Preservation of nerve graftsat -700C does not have any untoward effect.

During the last decades, autotransplantation tobridge peripheral nerve defects has become amethod of choice in most clinics. Usually cablegrafts are used of expendable sensory nerves andsutured interfascicularly as advocated by Millesi(1972). This technique minimises the tissue re-jection problem, and good results have been re-ported by Millesi (1972), and Samii and Kahl(1972). However, the following specifications foran ideal transplant remain unfulfilled:a. the normal neurolemmal system consisting ofa specific funicular arrangement (Sunderland,1968) of the parent nerve, which seems to be quiteimportant in achieving optimal qualitative andquantitative end results, is not replaced.b. the thickness and number of the transplantednerve segments are seldom the same as in theoriginal nerve involved.c. the available sensory nerves may be too shortto bridge a large gap in a thick nerve or gaps ofmore than one nerve at the same time.d. the sacrifice of the donated sensory nerve mightsometimes produce some neurological deficit inthe donor.Gutman and Sanders (1943) and Seddon and

Holmes (1944), in animal and human experimentswith homografts concluded that a graft of about30 mm long was the limit which could survive.Longer grafts showed diffuse inflammatoryreaction, leading to less than optimal bloodsupply and consequent fibrosis of the entire graft.This reaction was attributed to the immune reac-tion on the part of the host tissue, the severity andAccepted 13 January 1977

rapidity of which varied according to the geneticrelationship between donor and host.

Since that time many attempts have been madeto minimise the immunological reaction in labora-tory and in clinical applications. Das Gupta (1967),with electronmicroscope studies, showed that allo-genous nerve when transplanted is subjected to arejection process similar to that which occurs inother tissues. Attempts to reduce donor nerveantigenicity by irradiation (Marmor, 1967) haveproduced disappointing clinical results, despiteearlier promising experimental studies (Marmor,1964). Pollard and Fitzpatrick (1973) have des-cribed the mechanisms whereby the immune pro-cess hinders regeneration. Improved regenerationhas been demonstrated through allografts in ex-periments with animals treated with immuno-suppressive agents (Pollard et al., 1973).The aim of the presen't study was to determine

the role of tissue typing on regeneration throughpreserved peripheral nerve allografts.

Material and methods

Thirty-six beagle dogs were tissue typed using themajor histocompatibility complex system (Ceppel-ini et al., 1967). Peroneal nerve was removed andpreserved in tissue culture medium 96 in triplesealed plastic bags, at a constant minimal tem-perature of -700C under sterile conditions, for aperiod of four to six weeks, before implantationinto other dogs. The standard microsurgical tech-nique was applied using X16 magnification andlOXO atraumatic monofilament nylon. Three

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R. Singh, K. Mechelse, and S. Stefanko

series of experiments were carried out with differ-ent types of peroneal nerve transplants, in eachof which there were six compatible and six in-compatible dogs.Series 1: 40 mm peroneal nerve transplant sub-mitted to radiation at dry ice temperature(Marmor, 1964).Series2: 40 mm peroneal nerve transplant-noirradiation.Series3: 70 mm peroneal nerve transplant-noirradiation.About seven to eight months after the trans-

plantation, the nerve was re-exposed, the reactionto direct electric stimulation recorded, and thenerve conduction velocity determined, withanimals under general anaesthesia and usingbipolar platinum electrodes. Four electrodeswere placed at fixed distances of 20 mm along thenerve, the first proximal to and the last distal tothe graft. The compound action potential of theanterior tibial muscle was recorded through aconcentric needle electrode. The nerve was stimu-lated supramaximally with a square wave of 0.3 msduration. The temperature of the nerve rangedbetween 27°-300C. The maximum conductionvelocity of the graft was estimated from thelatency of responses of the anterior tibial musclefrom stimuli at the proximal and distal stimulatingpoints, and the conduction distance. In normaldogs the maximum conduction velocity of theperoneal major nerves ranged from 30 rn/s to50 mr/s.

According to the nerve conduction velocity,results were grouped into four grades:Grade I normal conduction velocity

>30 m/sGrade II reduced conduction velocity

20-30 m/sGrade III markedly reduced conduction velocity

15-19 m/sGrade IV no response.The transplanted nerve along with 10 mm of

proximal and distal end of the host nerve wasremoved for histological study. Longitudinal sec-tions at proximal and distal segments of the hostnerve were prepared with specific stains. To evalu-ate the degree of axonal regeneration, the Bodianmethod was employed. Kluever's method was usedto visualise the late stage of myelin regeneration.Special attention was paid to qualitative picturesof the nerve sheaths. The Van Gieson and haema-toxylin eosin method was used to visualise theconnective tissue.To make differential evaluation easier, care was

taken to obtain nerve sections at the same levelin relation to proximal and distal nerve suture

sites. A diagramatic representation shows theexact site of different longitudinal and transversesections in relation to the transplant (Fig. 1).

vL.__ -. _J- 1

prox.distal graft

Fig. 1 Diagram of sites of longitudinal andtransverse section (trans).

The quantitative and qualitative histologicalfindings were grouped into four grades:Grade I (Fig. 2a, b, c) good regeneration with be-ginning of myelinisation; parallel bundles of nervefibres; no fibrosis; no inflammatory reaction.Grade II (Fig. 3a, b, c) good regeneration; littleirregularity in parallel run of the nerve fibrebundles; slight fibrosis; no inflammatory reaction.Grade III (Fig. 4a, b, c) incomplete regeneration;slight interlacing of the nerve fibres; evidentfibrosis; slight inflammatory reaction.Grade IV (Fig. 5a, b, c) poor regeneration; markedinterlacing of nerve fibres; extensive fibrosis; evi-dent inflammatory reaction.For both EMG and histology, grade I was inter-

preted as the best and grade IV as the worst,grades II and III being intermediate. Results thusobtained were subjected to statistical analysis usinga nonparametric ranking procedure (Wilcoxon'stwo sample test) for comparison of the differentgroups.The neurological examination of the dog, which

in itself is not very reliable and could bias theinterpretation of the end results, was not takeninto consideration when summarising the endresults.

Results

SERIES 1(40 mm grafts, submitted to radiation)In the compatible group nerve conduction velocitywas normal in three cases and reduced in two.One dog was lost during the follow-up period. Inthe non-compatible group no response was re-corded in three cases, markedly reduced conduc-tion velocity in two, and reduced conductionvelocity in one case only.

Differences in the results of these two groups areof statistical significance (Fig. 6, P<0.01). Onhistological analysis four of the compatible groupwere graded as grade I and one grade II, in

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Role of tissue typing on preserved nerve allografts in dogs

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Fig. 2 Grade I of histological gradinig. (a) Site ofnerve suture. Slight lymphocytic reaction close to thesuture. (H & E X235). (b) Distal nerve segment.Regular course of Bunger-Hanke's strands(proliferation of Schwann cells). (H & E X380).(c) Distal nerve segment. (Bielschowsky stain). Densetexture of regenerated fibres (X 380).

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Fig. 3 Grade II of histo!ogscal grading. (a) Site ofnerve suture. Remnants of suture and polymorphinflammatory reaction with foreign body giant cells.(H & E X380). (b) Distal nerve segment. Slightirregularity of nerve fibre bundles. (BielschowskyX235). (c) Distal nerve segnment. Longitudinal courseof nerve fibres with uneven texture of buntdles.(Bielschowsky X235).

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Fig. 5 Grade IV of histological grading. (a) Site ofnerve suture. Marked interlacing of nerve fibres.Considerable proliferation of connective tissue andaberrant fibres. (Van Gieson X235). (b) Distal nervesegment. Diffuse inflammatory reaction in epi- andperi-neurium. (H & E X235). (c) Distal nervesegment. Poor regeneration (B.elschowsky X380).

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nerve conduction velocity was recorded in four,markedly reduced in one and no response at all inone case. On statistical analysis the difference inthese two groups, however, is not significant (Fig.8, P>0.05). On histological study, among the

3 -

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Ill IV grade III IV

6 EMG radiation +deep freezing 40 mm P<0.01.

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contrast to the non-compatible group where allsix were graded III and IV. This difference is alsostatistically significant (Fig. 7, P<0.01).These results indicate that better regeneration

was found in the compatible group of dogs thanin the non-compatible. On histological examina-tion there was marked foreign body and inflamma-tory reaction in the non-compatible group whilein the compatible group a more normal patternof regeneration, with minimal foreign body reac-

tion was found.Preservation of the nerves under these condi-

tions seems to have no adverse effect on the re-

generation of the nerve grafts. However, it isdifficult to estimate by this examination how farirradiation had been responsible for diminishingthe immune reaction of these transplanted nerve

grafts. For this reason series 2 was carried out.

SERIES 2

(40 mm grafts without irradiation)In the compatible group nerve conduction velocitywas normal in two cases and reduced in four. Inthe non-compatible group a normal conductionvelocity was not recorded in any case. Reduced

number6

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compatible non-compatible

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I I Il IV grade 11 III IV

8 EMG deep freezing 40 mm P>0.05.

compatible group four showed regeneration with a

very normal pattern (grade I), and one showedgrade II. In one case the suture line was found tobe broken (probably due to technical error), andthis was graded as IV. In the non-compatiblegroup none was good enough to be included ingrade 1, two were in grade II, three in grade I1I,and one in grade IV. Thus here also the results inthe compatible group seem a little better than inthe non-compatible group, although some of thegrafts in the non-compatible series did show some

definite signs of regeneration. Better qualitativeand quantitative regeneration along with minimalforeign body and inflammatory reaction was seen

in compatible cases compared with the non-com-

patible cases. The histological difference betweenthese groups is of statistical significance only if thecase where the suture line was found to be brokenis not taken into consideration. Otherwise thedifference is not significant (Fig. 9, P>0.05). There

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Fig. 9 Histology deep freezing 40 mm P>0.05.

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4- compatible non-compatible

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is no significant difference (P>0.05) in con-ductivity or histological grading of these 40 mmgrafts with and without radiation in compatibleand non-compatible series. However, a better typeof regeneration with less foreign body and inflam-matory reaction was found in cases where no

radiation was carried out, so radiation appears tobe without additional advantage.

SERIES 3

(70 mm grafts without irradiation)On EMG examination in the compatible group,five showed normal nerve conduction velocity,while the one where the suture line was found tobe broken registered no response at all. This is inmarked contrast to the non-compatible group

where no response could be recorded in threecases and only a response with markedly reducedconduction velocity in the remaining three cases.

The difference in these results is statistically sig-nificant (Fig. 10, P<0.05).

number61

Icompatible non-compatible

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I III IV grade I III IV

Fig. 10 EMG deep freezing 70 mm P<0.05.

On histological examination four from the com-

patible group were grade I and one grade II. Thecase with the broken suture line was grade IV. Inthe non-compatible group all showed marked in-flammatory, foreign body reaction, and fibrosiswithout any normal regeneration, and are thus in-cluded in grade IV. This difference between thetwo groups is also of statistical significance (Fig.11, P<0.05). Comparing these EMG and histo-logical results with those of 40 mm grafts withoutradiation, the difference in the non-compatiblegroup is statistically significant (P<0.05).

Discussion

These results support the view of Seddon andHolmes (1944) and Spurling et al. (1945) that someof the grafts of about 30 mm length might showsome signs of regeneration in spite of all the odds.

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1 III IV grade 11 III IV

Fig. 11 Histology deep freezing 70 mm P<0.05.

Some vascularisation of the graft could takeplace from either end of the host nerve. Longergrafts may have difficulty in surviving under ad-verse conditions. Ducker and Hayes (1970) sup-ported these views by their experimental study inchimpanzees, and commented that irradiatednerve grafts are unlikely to be succesful whenlonger than 40 mm.Comparison of individual histological and

electromyographic gradings (Table) showed thata grading agreement exists in 28 cases out of 35(80%) with discrepancies in seven cases (20%). Intwo cases the EMG was graded higher than histo-logy, and in five cases histology was graded higherthan EMG. Thus the shift was not of statisticalsignificance. Therefore, it can be concluded thatreasonable correlation exists between the histo-logical and electromyographic findings.

Table Correlation between histology and EMG

EMG grading

Grading Histology I II III IV

Grade 1 12 10 2 - -Grade 11 5 - 5 -

GradeIII 7 - 2 5 -

GradeIV - 2 1 8

Total number 35; grading agreement exists in 28 cases (80%).Discrepancies exist in seven cases (20%) (in two cases EMG gradedhigher than histology, and in five cases histology graded higher thanEMG).Conclusion: shift not significant (according to sign test).

These results can be compared with those re-ported by Samii et al. (1971) where an experi-mental study was carried out comparing autograftswith different types of nerve homografts in rabbitsbut the length of the graft used was not men-tioned. According to their study 75% of theanimals with autogenous grafts regained normalfunction in comparison with 33% which hadhomografts. In this experiment no emphasis has

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been laid on the return of neurological function,but even in 70 mm long preserved nerve allograftswith perfect tissue compatibility, a normal patternof regeneration was found in about the same per-centage of cases.

References

Ceppelini, R., Curtoni, E. S., Maltiuz. P. Z., Miggiano,V., Seudeller, G., and Serr, A. (1967). Genetics ofLeukocyte A ntigens. Histocompatibility Testing.p. 149, Munksgaard: Copenhagen.

Das Gupta, T. K. (1967). Mechanisms of rejection ofperipheral nerve allografts. Surgery, Gynecologyand Obstetrics, 125, 1058-1068.

Ducker, B., and Hayes, G. J. (1970). Peripheral nervegrafts; experimental studies in the dog and chim-panzee to define homograft limitations. Journal ofNeurosurgery, 31, 236-243.

Gutmann, E., and Sanders, F. K. (1943). Recovery offibre numbers and diameters in the regeneration ofperipheral nerves. Journal of Physiology, 101, 489-518.

Marmor, L. (1964). Regeneration of peripheral nervesby irradiated homografts. Journal of Bone and JointSurgery, 46A, 383-394.

Marmor, L. (1967). Peripheral Nerve Regenerationusing Nerve Grafts. Charles C. Thomas: Spring-field, Illinois.

Millesi, H. (1972). The interfascicular nerve-graftingof the median and ulnar nerves. Journal of Boneand Joint Surgery, 54A, 727-750.

Pollard, J. D., and Fitzpatrick, L. (1973). A com-parison of the effects of irradiation and immuno-suppressive agents on regeneration throughperipheral nerve allografts: an ultrastructural study.Acta Neuropathologica (Berlin), 23, 166-180.

Pollard, J. D., Mcleod, J. G., and Gye, R. S. (1973).Regeneration through peripheral nerve allografts.An electrophysiological and histological study fol-lowing the use of immunosuppressive therapy.Archives of Neurology (Chicago), 28, 31-37.

Samii, M., and Kahl, R. L. (1972). Klinische Resultateder autologen Nerventransplantation. MelsungenMedizinisch Mitteilungen, 46, 197-202.

Samii, M., Schurmann, K., Scheinpflug, W., andWallenborn, R. (1971). Experimental studies com-paring grafting with autogenous and irradiatedfreeze dried homologous nerves. Proceedings of theGerman Society for Neurosurgery, 22nd AnnualMeeting Dusseldorf. November 14-17. ExcerptaMedica: Amsterdam.

Seddon, H. J., and Holmes, W. (1944). Late conditionof nerve homografts in man. Surgery, Gynecologyand Obstetrics, 79, 342-351.

Spurling, R. G., Lyons, W. R., Whitcomb, B. B.,and Woodhall, B. (1945). Failure of whole homo-genous nerve grafts in man. Journal of Neuro-surgery, 2, 79-101.

Sunderland, S. (1968). Nerves and Nerve Injuries.pages 26 and 702. Churchill Livingstone: Edinburghand London.

Wilcoxon, P. (1973). In Statistical Methods in MedicalResearch. Edited by P. Armitage. p. 398. Blackwell:Oxford.

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