Transplantation into the human brain: present future possibilities - … · 6-OHDA lesioned rats.'34 Amphetamine-induced rotational asymmetry can be reversed by grafts re-innervating

Journal ofNeurology, Neurosurgery, and Psychiatry. Special Supplement 1989:39-54

Transplantation into the human brain: present status

and future possibilitiesOLLE LINDVALL

From the Department ofNeurology, University Hospital, and Department ofMedical Cell Research, University

ofLund, Lund, Sweden

Transplantation of brain tissue into the adult mam-malian CNS is not a new field of research. A centuryago, the New York Medical Journal contained anarticle by W Gilman Thompson' entitled "Successfulbrain grafting". He exchanged large pieces of neocor-tical tissue between adult cats and dogs and analysedsome of the grafts microscopically after survival timesofup to 7 weeks. Despite the positive title ofhis paper,the long-term "grafts" probably consisted only ofneuron-free remnants and developing scar tissue.Almost 90 years passed before the first animalexperimental data appeared clearly indicating thepossible clinical usefulness of the neural graftingtechnique. In 1979 it was shown23 that intracerebralgrafts of rat fetal dopamine (DA) neurons were able toreduce the symptoms of experimental, 6-hydrox-ydopamine (6-OHDA)-induced Parkinsonism inrats. This was the first example of neurons, beingimplanted into the adult mammalian brain, whichwere able to reverse a functional deficit in an animalmodel of a human neurological disorder. Early on, ofcourse, these findings kindled a hope that it would bepossible to develop a transplantation therapy forpatients with Parkinson's disease.

Since then, experimental data obtained in rats havesuggested the clinical application ofthe neural graftingtechnique also in other disorders. In experimentalHuntington's disease, induced by intrastriatal injec-tions of an excitotoxin (kainic acid or ibotenic acid),grafted fetal striatum is anatomically integrated intothe host brain4 and reduces both motor and cognitivedeficits.' In two animal models of dementia (youngadult rats with fimbria-fornix transections and cog-nitively impaired aged rats) grafted fetal cholinergicneurons from the septum-diagonal band region growinto the host hippocampus and amelio rate deficits inmemory and learning.v" Furthermore, fetal

Based on the Sir William Macewen Centennial Lecture, Society of BritishNeurological Surgeons, Glasgow, 23 September 1988.

Address for reprint requests: Olle Lindvall, M.D., Ph.D., Department ofNeurology, University Hospital, S-221 85 Lund, Sweden.

Accepted March 1989

noradrenergic neurons from the locus coeruleusregion implanted into the hippocampus suppressseizure development in hyperexcitable, previouslynoradrenaline-depleted rats subjected to electricalkindling2 13 (an experimental model ofcomplex partialepilepsy in humans).

However, the transition from the first positiveanimal experimental data to trials in patients shouldbe approached with caution. From the clinical point ofview, research in neural transplantation has by farreached the furthest in Parkinson's disease. Whetheror not intracerebral neural grafting will be successfulin patients with Parkinson's disease will be of greatimportance for the future application of this techniquein other disease states. Therefore, this review will focuson research in Parkinson's disease and summarisespresent experience with grafting in patients as well asits animal experimental basis. Finally, some issues willbe discussed that are of critical importance in order toestablish neural transplantation as a real therapeuticalternative in Parkinson's disease and otherneurological disorders.

Grafting experiments in animals-fetal substantianigraMost basic experimental studies have been carried outin rats with unilateral, 6-OHDA-induced lesions ofthemesostriatal DA system. Such rats exhibit a hemipark-insonian-like syndrome with the following maincharacteristics: the animals have an asymmetric pos-ture and display rotational asymmetry towards thelesioned side both spontaneously and in response toamphetamine (which releases DA from the intactmesostriatal system). Apomorphine, which stimulatessupersensitive DA receptors in the denervatedstriatum, causes rotation towards the non-lesionedside. The rats also show sensory inattention towardsstimuli applied to the side of the body contralateral tothe lesion.

Mesencephalic DA-rich grafts implanted directlyinto or adjacent to the 6-OHDA denervated striatumin the rat can reduce the lesion-induced motor andsensorimotor asymmetries.23 1415 The degree of

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behavioural recovery has been found to be related tothe extent of the transplant-derived DA reinnervationof the previously denervated striatum."'6 The grafttissue, which is taken from the ventral mesencephalonof 13 to 15 days old rat fetuses, can be implanted intoadult rats using two main procedures: (1) solid piecescan either be put in the lateral ventricle or intopremade cortical cavities on top of or lateral to thestriatum;23 161" (2) a suspension of cells can be injectedstereotaxically directly into the striatum.'" Theimplanted DA neurons grow into the denervated hoststriatum forming a terminal pattern similar to theintrinsic innervation.'9 They also establish synapticcontacts with host striatal neurons2"22 and receiveafferent inputs from the host brain.22"24 The grafts aremetabolically2" and physiologically3' active; theyexhibit transmitter synthesis, normal firing patterns,and spontaneous DA release. In the host striatum, thegraft-derived innervation causes post-synapticdopaminergic binding sites to return to normal den-sity32 and normalises firing rates and drug sensitivity ofhost striatal neurons.33

Furthermore, DA neurons implanted into specificsubregions of the denervated striatum compensate forspecific features of the hemiparkinsonian syndrome in6-OHDA lesioned rats.'34 Amphetamine-inducedrotational asymmetry can be reversed by grafts re-innervating the dorsal caudate-putamen, whereasgrafts innervating the ventrolateral part of thestriatum ameliorate deficits in sensorimotor attentionbut have no effects on rotational asymmetry. Thefunctional recovery is only obtained if the DA-richmesencephalic grafts are placed near the target area,that is, within or directly outside the striatum.Implantation ofDA neurons into the substantia nigra,the actual site of cell body loss after the 6-OHDAlesion, or along the trajectory of the nigrostriatalpathway, has not led to the behavioural effects accom-plished with grafts localised in the striatum.33 This ismost probably due to the inability of the grafted DAneurons to extend their axons over a great distance tothe forebrain target region.The functional effects are clearly dependent on the

survival and continuous presence ofthe graft, since ifitis removed or destroyed at any time after transplanta-tion, the deficits immediately reappear.'6366 3 The graft-induced recovery from motor asymmetries is specificfor DA-rich ventral mesencephalic tissue so thatintrastriatal grafts of tissue rich in 5-hydroxytryp-tamine-producing neurons (from the mesencephalicraphe region) or of tissue appropriate to the target(from the striatum) do not lead to any functionalcompensation.37Although several motor and sensorimotor deficits in

6-OHDA lesioned animals can be ameliorated by DA-rich intrastriatal grafts, other behavioural abnor-

Lindvallmalities have proved resistant to graft-inducedrecovery. These include deficits in hoardingbehaviour3" and the aphagia and adipsia seen afterbilateral 6-OHDA lesions39 40 as well as the impairmentin the use of the contralateral forelimb for discreteskilled movements after unilateral lesions of themesostriatal DA system." It has been proposed (see,for example, ref41) that the differences in the effects ofgrafts on different behavioural measures reflect thedegree of incorporation of the graft into the hoststriatal circuitry. In their ectopic striatal location,probably only part of the anatomical and functionalconnections characteristic of normal mesostriatal DAneurons are re-established by the grafts. The differen-tial sensitivity of different aspects of behaviour tograft-induced recovery in the 6-OHDA lesioned ratcould have important clinical correlates. Nigral graftsmay be effective for some but not for all symptoms ofParkinson's disease in patients. Modifications of thetransplantation procedures or priming of the graftspharmacologically38 might be required to overcomethese limitations.

Successful grafting into the striatum of DA-rich,ventral mesencephalic tissue from fetuses has alsobeen reported in non-human primates with MPTP-induced Parkinsonism. Survival of implanted DAneurons in the caudate nucleus or the putamen hasbeen demonstrated microscopically in rhesus mon-keys,'2" African green monkeys4-"' and marmosets.48Biochemical data' have indicated that the grafteddopaminergic neurons are able to normalise DAturnover in previously severelyDA depleted areas alsoin non-human primates. Some of the studies havereported a long-lasting reduction of the MPTP-induced motor abnormalities, including hypokinesia,rigidity and tremor.

Grafting experinents in animals-adrenal medullaSoon after the initial reports of functional effects offetal DA neurons in experimental Parkinsonism (seeabove), alternative sources of catecholamine-produc-ing cells suitable for grafting were also being explored.At that time it seemed unclear whether it would ever bepossible, mainly for ethical and immunologicalreasons, to use human fetal tissue for transplantationin man. Although chromaffin cells from the adultadrenal medulla have by far attracted the greatestinterest, animal experiments have suggested that othertissues could also be potentially useful for grafting inParkinson's disease patients, for example, sympatheticganglia,5" 3 carotid body glomus cells52 and cell lineslike PCl2.52"-1Adrenal medulla cells can be implanted into the

lateral ventricle5' " or the DA-depleted striatum ofratsor monkeys.5"' The number of surviving chromaffincells is, however, low both in rodents52 and particularly


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Transplantation into the human brain: present status andfuture possibilitiesin monkeys.596263 Normal chromaffin cells secreteprimarily adrenaline and only low amounts of DA.'In long-term intraventricular adrenal medullarygrafts, DA and noradrenaline are the predominantcatecholamines,65 thus suggesting that transplantationcauses a shift in the relative amounts of catecho-lamines produced by the adrenal medulla. There isalmost no fibre outgrowth from the implantedchromaffin cells, which is probably due to inadequateactivity of nerve growth factor (NGF) in the adultstriatum. Addition ofNGF to the graft, preferably viacontinuous infusion through a dialysis fibre for severalweeks, significantly increases cell survival, causes atransformation ofmany cells towards a more neuronalphenotype and greatly enhances the fibre outgrowthinto the host striatum.i Survival of adrenal medullarycells has been demonstrated in the rat for at least 17months after intrastriatal grafting even without addi-tion of NGF,58 but there appears to be a need for acontinuous supply of NGF to maintain the graft-derived fibre outgrowth in the host striatum.58The functional capacity of adult adrenal medulla

cells implanted either into the striatum or the lateralventricle has only been demonstrated as an ability toreduce drug-induced rotational asymmetry in hemi-parkinson rats or monkeys. In unilaterally 6-OHDA-denervated rats, adrenal chromaffin grafts reduceapomorphine-iandpossiblyalsoamphetamine-inducedturning response.52 568 The adrenal grafts do notrestore contralateral sensorimotor inattention6' andno effect on spontaneous rotational behaviour (exceptduring the first hours after implantation) has beenreported. The reduction of apomorphine-inducedrotational asymmetry is stable up to 3 months aftergrafting but then disappears. However, if NGF isadded to the graft through continuous infusion eitherinto the striatum or into the lateral ventricle, thefunctional effect (as assessed by the apomorphinerotation test) is clearly increased58" and more long-lasting.58 With intrastriatal infusion of NGF in thevicinity of the grafts for 4 weeks after implantation,apomorphine-induced rotational behaviour was sig-nificantly reduced also at 6 and 12 months aftergrafting.58 Whether this higher functional potency ofadrenal medulla grafts supplied with NGF is due tograft differentiation and neurite outgrowth, as sugges-ted by Stromberg et al,58 can however, be questionedon basis of the recent report by Pezzoli et al.' Theyfound that NGF plus non-catecholamine-producingtissues (sciatic nerve or fat) were as effective as NGFplus adrenal medulla to reduce apomorphine-inducedrotational asymmetry in unilaterally 6-OHDA dener-vated rats.The very sparse information reported from

experiments with adrenal medulla transplantation innon-human primates is in good agreement with the

data obtained in rodents. Bankiewicz et al63 implantedadrenal medullary tissue into the DA-denervatedcaudate nucleus of unilaterally MPTP-treated rhesusmonkeys. Apomorphine-induced rotational asym-metry was attenuated for 3 months after surgery butby 6 months it had returned to control levels. Whetherdeficits in spontaneous behaviours can be reduced byadrenal medullary grafts in non-human primates isunclear since both improvement of the use of theaffected arm in hemiparkinsonian monkeys63 67 as wellas no effect on this parameter4449 have been describedafter surgery.

Grafting experiments in animals-comparison betweenfetal substantia nigra and adrenal medullaThe available data from basic experimental graftingstudies using either ventral mesencephalic tissue oradrenal medulla cells clearly favour the strategy withfetal DA neurons in clinical trials. There seems to bethree major advantages: First, although both types ofcells can improve deficits in rotational behaviourapparent after administration of apomorphine oramphetamine, only nigral cells have been convincinglydemonstrated to reduce abnormalities in spontaneousbehaviours. These include spontaneous rotationalasymmetry, sensory inattention and akinesia in 6-OHDA-lesioned rats and hypokinesia, rigidity andtremor in MPTP-treated monkeys. From the clinicalpoint of view, the capacity of the graft to improvedeficits in spontaneous behaviour probably is ofcritical importance in order to produce relief ofsymptoms in patients with Parkinson's disease.Second, in animals with 6-OHDA or MPTP-inducedParkinsonism the fetal nigral grafts show long termsurvival and the functional effects are permanentunless the graft is removed. In contrast, the reductionof rotational asymmetry seen after implantation ofadrenal medulla is transient and the chromaffin cellsshow atrophy unless NGF is supplied.Third, the fetalDA neurons form appropriate synapticcontacts with the host striatum and restore a well-controlled DA neurotransmission. Adrenal medullacells, on the other hand, possibly operate via a diffuse,non-controlled release of catecholamines (see,however, ref 68). This difference could be of criticalimportance for the usefulness of the grafts in patientswith Parkinson's disease.

Grafting experiments in humans-adrenal medullaDespite the encouraging animal data obtained withfetal DA neurons, the first clinical trials with trans-plantation in Parkinson's disease were performedusing the patient's own adrenal medulla to avoid theethical and immunological problems linked to the useof human fetal tissue. Between 1982 and 1985 fourpatients with severe Parkinson's disease were sub-

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42jected to adrenal medulla autotransplantation using astereotaxic procedure69'0 (fig 1). Two of them weregrafted in the caudate nucleus and two in the putamen.No adverse effects of the transplantation wereobserved. There was a minor improvement of motorfunction lasting for about 2 months but at 6 months nopositive effects remained. The long-term follow up ofthese patients has not indicated any influence of theadrenal medulla autotransplantation on the naturalcourse of the disease.The results from these trials, which did not give

anything of long-lasting therapeutic value to thepatients, supported the idea that the symptoms ofParkinson's disease can be improved, seemingly withvery little risk for the patient, through implantation ofcatecholamine-producing cells in the basal ganglia. Itseemed clear, however, that the transplantationprocedure had to be improved before any furtherclinical trials could be carried out.

In April 1987 Madrazo and co-workers reported thefirst successful adrenal medulla autotransplantationsin two young patients with Parkinson's disease."Instead of a stereotaxic approach Madrazo andcoworkers used open microsurgical techniques andthrough the cerebral cortex implanted pieces ofadrenal medullary tissue into a premade cavity in thehead of the caudate nucleus (fig 1).

After Madrazo's report was published the numberof Parkinson's disease patients subjected tointracerebral grafting increased rapidly (fig 2).Whereas only single cases were operated in 1982 to1985, about 15 patients received implants in 1986 andabout 100 patients in 1987 and 1988. In the majority ofpatients the adrenal medullary tissue has been implan-ted into a cavity in the caudate nucleus according toMadrazo et al,7 but in a few cases successful attemptsto use stereotaxic surgical techniques have been repor-ted.72 Madrazo and co-workers have summarised73their findings from the first 22 patients subjected toadrenal medullary autotransplantation. Four of thosepatients have died due to cerebral venous thrombosis,bronchial aspiration, acute necrotic pancreatitis, andmassive pulmonary thromboembolism, respectively.73Eighteen have been followed clinically for more thanone year postoperatively. According to the scoringsystem of Madrazo et al73 nine of the patients had apoor and nine patients a moderate performance beforesurgery but after transplantation 13 patients had agood, four a moderate and one patient a poorperformance. From these data it can be concluded thatat least about 80% of Madrazo's patients haveimproved after surgery. The improvement has beendescribed as a persistent reduction (at least up to 27months) of tremor, rigidity and akinesia both duringon and off periods. The daily doses of levodopa havebeen reduced by about 57% and two patients are

Lindvall

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STEREOTAXICSURGERY

" PATIENT'S OWN) ADRENAL GLAND

PIECES OF CHROMAFFINTISSUE

OPENMICROSURGERY

Fig 1 Schematic representation of the two alternativeprocedures usedfor grafting ofadrenal medulla cells into thestriatum ofpatients with Parkinson's disease. Pieces ofchromaffin tissue are either implanted into the caudatenucleus or putamen using stereotaxic surgical techniques orwith open microsurgery into a premade cavity in the head ofthe caudate nucleus.

without levodopa medication. Madrazo and co-work-ers73 have reported that in the 18 surviving cases thecomplications of the procedure were transient anddisappeared within one to two weeks. They includedpsychiatric disturbances with hallucinations, stupor,delusions, perseveration, respiratory problems, andpulmonary thromboembolism. The one exception wasa patient who sustained damage to the fornix duringoperation and who had memory impairment.About 250 operations using adrenal medullary

autotransplantation have been carried out in severalcountries but mainly in Mexico and the United States.The scientific reports from these trials are very few and


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Transplantation into the human brain: present status andfuture possibilities

TRANSPLANTATION INPARKINSON S DISEASE

1982 1983 1984 1985 1986 1987 1988YEAR

Fig 2 Estimated numbers of Parkinson's disease patients subjected to intracerebral grafting ofcatecholamine-producing cellsinto the striatum. In most cases the implanted tissue was the patient's own adretal medulla but about 50 patients have receivedgrafts ofhumanfetal mesencephalic tissue.

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the available data mainly derives from the experiencein the United States. From the preliminary results ofabout 90 operations recently reported by differentgroups in the United States (Third United ParkinsonFoundation Workshop on Brain Implants, Chicago,November 1988), it can be stated that only about 40%of the patients show some improvement. It has notbeen possible to reproduce the dramatic improve-ments reported by Madrazo and co-workers.7 73 In adetailed analysis7475 of severely disabled Parkinson'sdisease patients followed for six months after adrenalmedulla autotransplantation, performed according tothe technique ofMadrazo et al,7' a modest reduction ofthe duration and severity of "off" periods wasobserved. The mean percentage of "on" time duringthe day increased from 48% before transplantation to75% at six months after surgery. Even if the patientswere more independent during "off>' periods, theyremained prominently affected by their disease. The

dosages of antiparkinsonian drugs could not bedecreased after transplantation.When evaluating the relative value of this procedure

it is ofcourse also important to note that the mortalityrate, as well as the serious morbidity rate, as estimatedfrom the results presented by different groups in theUnited States, is as high as 5 to 10%. A majority ofpatients show postoperative psychiatric and respi-ratory disturbances.74 Furthermore, it is a seriousdrawback with this procedure that the mechanisms ofimprovement are largely unknown. As will be discus-sed below, it seems unlikely that the reduction ofParkinsonian symptoms is due to catecholaminerelease from the implanted cells as originally intended.It is furthermore not clear whether survival of the graftis necessary. In conclusion, open microsurgicalautografting of adrenal medulla must be regarded asan experimental approach and not an establishedtreatment for Parkinson's disease.

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Grafting experiments in humans-fetal substantianigraOn the basis of our own experience with adrenalmedulla autotransplantation in patients6"0 as well asthe more solid data obtained with grafting offetal cellsin experimental Parkinsonism (see above), a pro-gramme was initiated in Sweden in 1985 in order toapply the neural grafting technique in patients withParkinson's disease. In 1985 the ethical question wasdebated in the Swedish Society of Medicine and thisled to the adoption in 1986 of provisional ethicalguidelines for the use of human fetal material fortransplantation purposes. In summary, according tothese guidelines women undergoing abortion mustgive their consent before tissue material, taken fromthe dead fetuses, can be used for transplantation. Thetransplantation cannot in any way influence how,when and why the abortion is carried out. Noconnection between a particular donor and a recipientis allowed.

In a series of experiments directly related to theclinical application of the neural grafting technique inParkinson's disease, human fetal DA neurons wereimplanted into the DA-denervated striatum ofimmunosuppressed rats."'0 The optimal donor agewas found to be eight to 10 gestational weeks (fig 3Aand B), and ventral mesencephalic tissue from fetusesof these ages (but not from older ones) survivedtransplantation and had functional effects. The DAcells reinnervated the entire rat striatum forming aterminal pattern resembling the intrinsic one (fig 4A-E) and with synaptic contacts with host striatalneurons (fig 5A, B). The human DA-rich graftsspontaneously released DA and reversed deficits inboth drug-induced and spontaneous rotational

Lindvallbehaviour (fig 6). The ventral mesencephalon wasintact and could be used for implantation into aParkinsonian patient in about 50% of the abortions,which were carried out by suction (the abortiontechnique used for the fetal ages optimal for DAneuron survival). Repeated cultures showed that theentire procedure could be carried out without bacterialcontamination.

In November and December 1987 two 50 year oldwomen with Parkinson's disease since 1973 receivedintrastriatal grafts of human fetal DA neurons (formore details see ref 81). Using stereotaxic surgicaltechniques (fig 7) ventral mesencephalic tissue fromfour 8-10 week-old fetuses were implanted as a cellsuspension on the side contralateral to that with themost severe symptoms of Parkinson's disease. Thesuspension was implanted at two sites in the putamen(tissue from one fetus in each) and at one site in thecaudate nucleus (tissue from two fetuses). The patientswere put on immunosuppressive treatment at the timeoftransplantation using standard regimens ofcyclosp-orin, azathioprine and prednisolone. The same dosesof antiparkinsonian drugs were given during the sixmonths preceding the transplantation and during thesix months follow-up period.The results from up to six months postoperatively

can be summarised as follows.8' First, there has beenno marked changes in the percent time of the day thepatients spend in an "on" phase; Second, smallimprovements have been noted in clinical tests of themotor performance of both patients during offperiods. In patient 1 this slight bilateral improvementbegan at about three months after transplantation andwas more marked contralateral to the implantedside. The motor performance of patient 2 was more

Fig 3 (A) The dissected central nervous systemfrom a humanfetus obtained at a suction currettageabortion (9 weeks gestational age). In (B) the ventral mesencephalon (arrow-head) has beendissected out. tv = telencephalic vesicle, m = mesencephalicflexure, p = pontineflexure,c = cervicalflexure. Scale bar = 2 mm. From ref 78.


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Fig 4 (A-E) Light microscopic pictures ofsections, stainedfor tyrosine hydroxylase (TH) immunocytochemistry todemonstrate preswnedDA neurons,from an immunosuppressed rat receiving human fetal mesencephalic grafts (20 weekssurvival after implantation). (A) Overview ofa section through the mesencephalon showing the effects of the unilateral6-OHDA lesion. On the intact side, numerous DA neurons are seen in the substantia nigra (SN) and ventral tegmental area.On the side ofthe lesion virtually no TH-immunoreactivity is visible. (B) Overview ofa section through the grafted caudate-putamen, illustrating the size andposition ofa transplant (t;from a human fetus, 10-12 weeks gestational age) 20 weeks post-grafting. (C) Higher magnification ofa group ofpresumedDA neurons (arrows) situated within the human mesencephalicgraft illustrated in (B). The graft is also seen to have a very dense plexus of TH-positivefibres. Note that the photograph isoriented so that dorsal is to the right and medial is upward. (D) Higher magnification ofa bundle ofcoarse processes, probablydendrites (double arrow heads), which extend into the host striatumfrom the graft. (E) High magnification ofgraft-derivedfine-calibre TH-positivefibres (arrowheads) in the host caudate-putamen. Scale bars; (A) I mm; (B) 05 mm; (C) 75 pm;(D) 50 pm; (E) 15 plm. From ref 78.

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Fig 5 (A, B) Photomicrographs showing synapticspecialisations between grafted human DA neurons and hoststriatal neurons at 20 weeks post-grafting. In (A) aTH-positive boutonforms a symmetrical synapse (arrow)onto a dendritic shaft (d) and in (B) a largeTH-immunoreactive fibreforms a symmetrical synapticcontact (arrow) with a dendritic spine(s). Scale bars: (A)and (B) 0-25 gim. From ref 78.

variable, but at six months she performed the testsmore rapidly than preoperatively, with no obviousside to side difference. However, in neurophysiologicalmeasurements of simple and complex arm and handmovements patient 2 showed a significantimprovement on the side contralateral to transplanta-tion but not on the ipsilateral side. Third, the durationof the response to a single levodopa dose did notchange significantly in either patient; fourth, themotor readiness potential, which has been reported tobe diminished in patients with Parkinson's disease,82increased gradually during off in both patients; fifth,

Lindvallpositron emission tomography (PET) performed at 5-6 months postoperatively, did not show any increaseof 6-L-(18F)-fluorodopa uptake in the graftedstriatum.The lack of a major clinical improvement in these

patients can, in view of the PET data, most likely beascribed to insufficient number of surviving graftedDA neurons. While this could be due to more generalproblems discussed below (for example, scaling up,immunological rejection, adverse effects on graftedcells by the continuous antiparkinsonian medicationor the disease process itself), it also seems possible thattechnical factors such as the diameter of the implanta-tion instrument and the time interval between theabortion and implantation could have been of impor-tance.

Clinical trials with neural transplantation in Parkin-son's disease are also going on in Mexico,83 England,4USA, Spain, People's Republic of China, and Cuba.About 50 patients with Parkinson's disease have so farreceived intrastriatal implants of human fetal DAneurons. Both stereotaxic injection of a DA rich cellsuspension into striatum (see for example, ref 84; fig 7)and implantation of pieces of ventral mesencephalictissue into a cavity in the caudate nucleus (see ref 83; fig7) are being used. No detailed scientific reports fromthese studies have yet been published but the prelimin-ary descriptions of the results indicate in some casesmuch more marked improvements than was observedin the two Swedish patients.The reasons for the discrepancies between the

results obtained by different groups are unknown. Onemajor difficulty, at present, is that the procedures usedfor assessment of the patients' symptoms as well as thetime the patients have been followed preoperativelydiffer markedly between various studies. This meansthat the reported data are difficult to compare. Therehave been few attempts to demonstrate survival andingrowth of grafted DA neurons, for example, withPET. Thus, it is not known if the reportedimprovements are to any extent dependent on restora-tion ofDA transmission in the striatum as intended, ordue to some other mechanism (see below). It seemsunlikely that the early improvements, occurring withindays to weeks, reported by Madrazo et al83 andHitchcock et al,8' are due to a graft-derived reinnerva-tion of the striatum. There is in human-to-rat graftingexperiments a close correlation between the beginningof fibre ingrowth and synapse formation with hoststriatal neurons at about 2-3 months, and the onset offunctional effects.78 (fig 6).

Possible mechanisms ofaction ofcatecholamine-producing cell implantsA major problem when analysing the data reportedfrom the human trials is that graft survival and graft-


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FIBRE INGROWTH AND SYNAPSEFORMATION BEGIN HERE

PRE-TRANS-

PLANTATION

I I I10 15 20

WEEKS POST-TRANSPLANTATION

Fig 6 Relationship between the time course ofdevelopment offunctional effects and that ofDAfibre ingrowth and synapseformation after implantation of ventral mesencephalic tissuefrom a humanfetus into the DA-depleted striatum ofimmunosuppressed rats. The amphetamine-induced net rotation asymmetry scores (turns contralateral to the lesion sidesubtractedfrom turns ipsilateral to the lesion side) are shown both before and at various time points after transplantation for a

group ofunilaterally 6-OHDA denervated rats that received DA-rich implantsfrom a 9 week old human fetus.76 Morphologicaldatafrom ref 78.

induced reinnervation have not yet been convincinglydemonstrated in any of the operated Parkinson'sdisease patients. In the few cases that have beensubjected to autopsy (all implanted with adrenalmedullary tissue in the caudate nucleus according toMadrazo et al") either no surviving graft8"6 or a verylimited number of presumed adrenal medulla cells87(chromogranin A-positive but tyrosine-hydroxylasenegative) have been demonstrated. Studies performedwith PET in a few patients with grafts of adrenalmedulla' or fetal nigra8" have not detected any signs ofincreased catecholaminergic function in the striatumafter transplantation (as assessed by the uptake of 18-F-fluorodopa and l-C-nomifensine). It should bepointed out, though, that most of the cases analysed

have shown very little clinical benefit from the opera-tion (which agrees with a poor graft survival) and itcan therefore not be excluded that in the more

successful cases there is also a higher yield of survivingcatecholamine-producing cells. Unequivocal demon-stration of graft survival and reinnervation of the hoststriatum remains, however, one of the major scientificchallenges in future clinical trials with transplantationin Parkinson's disease.What are the possible mechanisms of action of

catecholamine-rich cell implants underlying theobserved improvements in patients with Parkinson'sdisease? If other explanations for the clinical changescan be excluded, such as placebo responses, or mani-pulations with antiparkinsonian drug treatment, there

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CELLSUSPENSION

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VENTRAL MESENCEPHALONFROM HUMAN FETUS

PIECES OF MESENCEFIHALICTISSUE

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STEREOTAXIC OPENSURGERY MICROSURGERY

Fig 7 Schematic representation of the two alternative procedures usedfor grafting ofhuman fetal DA neurons into thestriatum ofpatients with Parkinson's disease. The ventral mesencephalon is either prepared as a cell suspension which isimplanted stereotaxically into the caudate nucleus and/or putamen, or divided in pieces which are put into a premade cavity inthe head of the caudate nucleus.

are three major possibilities (fig 8A-C): First, thegrafts could work as biological minipumps and releaseDA (or other catecholamines) diffusely into thestriatum or into the cerebrospinal fluid (fig 8A). Thishas been reported to occur when rat adrenal medullacells are implanted into the rat striatum," in whichcase a leakage oftransmitter into the surrounding hosttissue can be demonstrated. Such a diffuse cate-cholamine release has been proposed to account forthe functional effects observed after adrenal medullaautotransplantation, at least in rats. This is supportedby the finding that chronic infusion of DA into thedenervated striatum using a mini-pump is able toreduce apomorphine-induced rotational asymmetryto the same degree as intrastriatal adrenal medullarygrafts.' Indeed, the first human trials were carried outwith the intention of utilising this mechanism also inthe Parkinsonian brain but so far there is littleevidence that the grafted chromaffin tissue works inthis way in patients except perhaps during a transientperiod.70 Analyses of catecholamines and theirmetabolites in ventricular cerebrospinal fluid follow-

ing adrenal medulla grafting according to Madrazo etal" have not demonstrated any significant increaseseven in patients who have exhibited clinicalimprovement."92

Second, the grafted cells may grow into the hoststriatum and restore synaptic neurotransmission (fig8B). This mechanism of action seems more likely withimplants of fetal neural tissue than after adrenalmedulla autotransplantation (except possibly in afuture clinical setting when NGF is administered tothe graft). There are, however, no PET data availableto support the idea that the improvements reportedafter grafting of fetal mesencephalon can be attributedto a graft-derived increase of dopaminergic nerveterminal density in the striatum. Furthermore, aspointed out above, the time course of theimprovements reported from the patients operated inMexico83 and England' differs markedly from that ofingrowth and synapse formation from human fetalDA neurons77 78 (at least when xenografted into the rat,fig 6).A thirdpossibility is that the graft, at least for a time,

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(A) (C)

DIFFUSE RELEASE TROPHIC ACTION

Fig 8 A-C. Schematic illustration of three possiblemechanisms ofaction ofcatecholamine-producing cellimplants in Parkinson's disease. In (A) the two grafts(chromaffin tissue) extendfew processes into the caudatenucleus andputamen of the host, and instead act via a difuserelease ofcatecholamines into the surrounding striatal tissue.In (B) the grafts (fetal mesencephalic tissue) have growninto the host striatum,formed a new terminal plexus, andreinstated synaptic neurotransmission. In (C) the grafts(chromaffin tissue) show very little outgrowth but havepromoted sprouting of the host's own DA system leading toan increased density ofDA terminals in the striatum.

exerts a trophic action on the host brain (fig 8C). Thismechanism, which does not require long-term survivalof the graft, could lead to sprouting of the fewremaining intrinsic DA neurons or stimulate otherrecovery mechanisms in the striatum ofthe Parkinson-ian patient. So far, no evidence has been provided thata trophic mechanism underlies the improvementsreported in patients either after transplantation offetal nigral cells, or adrenal medulla. However, recentstudies performed in MPTP-treated mice and mon-keys suggest that intrastriatal implantation of fetalnigra or adrenal medulla can indeed promote sprout-ing of remaining host dopaminergic neurons.49 6393 4This effect can at least partly be attributed to the

(B) parenchymal injury produced by the transplantationtechnique. In monkeys, adrenal medulla grafts placed

SYNAPTIC RELEASE either in a cavity63 4 or stereotaxically9 into the

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50caudate nucleus induced a presumed sprouting res-ponse ofDA fibres to an extent that corresponded tosurgical procedures without any tissue implants. Thesurvival of adrenal chromaffin cells in the grafts wasminimal.63"' According to Bankiewicz et al49 fetalnigral tissue is more effective and gives rise to extensivesprouting of presumed dopaminergic neurons in thehost brain.

Some critical issuesfor successful clinical applicationofthe neural grafting techniqueScaling up. Most experimental studies with neuraltransplantation have been carried out in rodents andonly a few in monkeys. In the rat striatum between 100and 200 surviving DA cells are necessary in order toeffect a greater than 50% reduction of amphetamine-induced rotational behaviour in hemiparkinsonianrats.95" This number of cells constitutes only about 1-2% of the neurons forming the intrinsic mesostriatalDA system in the rat.97 Given the rat data, what is thecritical number of surviving grafted DA neuronsneeded in the human brain to obtain a therapeuticeffect? Optimally, 20 000-25 000 DA cells from bothsides of the mesencephalon of each human fetus (8 to10 gestational weeks) survive grafting to the striatumofimmunosuppressed rats." Since it can be estimated98that the human putamen and caudate nucleus arenormally innervated by about 60 000 DA neuronseach, grafting of ventral mesencephalic tissue fromone fetus into one of these structures might be able torestore 30 to 40% of the normal number of cells. Thesymptoms of Parkinson's disease do not appear untilmore than 70% of the DA neurons have degen-erated9"'°; until this stage is reached, DA transmissionis believed to be maintained through hyperactivity ofremaining DA neurons and post-synaptic receptorsupersensitivity.'0' 102 It therefore seems realistic topostulate that tissue from one or more human fetuses,implanted unilaterally into the putamen, caudatenucleus or both, would be sufficient to effect somesymptomatic improvement for a patient with Parkin-son's disease.

It should be remembered, though, that the volumeof the putamen is 200 times larger in man than in therat brain. To what extent this structure will bereinnervated is not only dependent on the number ofsurviving grafted DA neurons but also on the numberand location ofimplantations and the growth capacityof each DA neuron. Grafted rat DA neurons extendtheir fibre network about 1 5-2 mm from the graftborder,'9 whereas human DA neurons implanted intothe rat striatum have exhibited a growth distance of atleast 3 mm," that is until the growing fibres reach theborder of the striatum. The human DA neurons thushave a higher growth capacity than rat DA neuronsbut the maximum extension of their axonal processes

Lindvallis not known. This is, ofcourse, ofgreat importance inorder to decide the number of implantation sites.Before we performed our first grafting experiments inpatients, we estimated that each human DA neuroncould grow up to 4a1 to 5 4 mm. Theoretically, about40 to 80% ofthe volume ofthe putamen would then bereached by the growing DA axons implanted alongtwo injection tracts. The lack ofany major effect couldin part be due to an overestimation of the growthcapacity ofhuman DA neurons and may indicate thata larger number of implantation sites are necessary.

Host environment. A common feature of theexperimental models of human neurodegenerativedisorders used in grafting experiments is that theexposure to the causative agent (for example, 6-OHDA, MPTP) occurs during a short period of timeand has ceased at the time of cell implantation. Inidiopathic Parkinson's disease the aetiology is un-known and it may therefore by hypothesised that thedisease process itselfcould interfere with the long-termsurvival and outgrowth ofDA neurons implanted intothe patient's brain. This would most likely not occur ifthe degeneration is due to an intrinsic defect in theirown DA system, such as a premature or acceleratedaging process'031 or a lack of scavenger moleculesthat can remove endogeneously produced freeradicals.'0'07 If, on the other hand, Parkinsonianpatients lose DA neurons because of a disease processwithin the striatum but extrinsic to these neurons, forexample to a lack of trophic substances, this processcould also elicit a degeneration of the grafted neurons.Similarly, if the DA neurons have degenerated due toan exogeneous insult, of which the causative agentwere still present, this process could also cause agradual death of DA neuron in the graft or couldinhibit growth. This would be the case if the patientcontinues to be exposed to some environmental toxin(see refs 108-110). However, if the degenerationresulting from this exposure is a slow process, onewould expect that the patient should still benefit fromDA neuron grafting, at least for a limited time period.

In severely affected Parkinson's disease patients,drug therapy must be maintained until beneficialeffects appear, which would be expected at about threemonths postoperatively. However, the graftedneurons may be vulnerable to this treatment, par-ticularly during the period of nerve fibre formation. Ithas been suggested that levodopa treatment mayaccelerate the degeneration of remaining intrinsicnigrostriatal DA neurons in Parkinsonian patients(see ref 111). This might occur, for example, throughan autoxidation of levodopa which has been reportedto produce many cytotoxic substances such as freeradicals and quinones."' One cannot rule out thepossibility that levodopa treatment interferes with thesurvival and growth of the grafted DA neurons; this


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Transplantation into the human brain: present status andfuture possibilitiespossibility should be tested in animal experiments.Immunosuppression. Although the brain is often con-sidered to be immunologically privileged (see refs 113and 114) it is now well established that immunologicalrejection processes occur also in the CNS.?6 15 116 Fetalneural tissue has been shown to be immunogenic"6117and can express major histocompatibility complex(MHC) antigens (the strong transplantation antigens)after the proper inductive stimuli. In fact, in vitroexpression of MHC antigens has been demonstratedafter intracerebral grafting. 116 There is evidence ofhostimmunisation by fetal neural grafts ofhuman origin ina xenograft situation." Immune reactions as well asfulminant rejections have been observed in animalrecipients ofhuman fetal neural tissue."7

However, implantation of allografts of fetal sub-stantia nigra into the brains of MPTP-lesioned non-human primates has in most cases been performedwithout immunosuppression. The available reportshave indicated good graft function up to at least sevenmonths postoperatively.47 8 Only one case of regraft-ing has been described in monkeys in which a promptloss of graft function was observed on the previouslyimplanted side."8 It is not known if immunosuppres-sion is necessary in clinical trials with grafting ofhuman fetal neuronal tissue, However, it seems atpresent appropriate to use such treatment in order tooptimise the condition for graft survival and toincrease the chance for successful regrafting. Ifimmunosuppression is necessary a major problem isthat, at present, there are no non-invasive approachesto monitor rejection ofgrafted fetal tissue in the brain.If it precedes any functional effects, rejection wouldprobably pass unnoticed. If positive functional effectshave developed, rejection would lead to a partial orcomplete disappearance of such graft-induced im-provements. In either situation it would be desirable todetect the first signs of a rejection process in the brainin order to be able to prevent the rejection of the graft.

Concluding remarksMuch experimental evidence, derived from severalanimal models, suggests that cell transplantation intothe adult CNS has a great potential to becomevaluable in the treatment of a variety of humanneurological disorders. Although grafting of fetalneurons seems to be the most promising approach atpresent, other sources of cells, such as adrenal medullaor cultured cell lines, may provide alternativestrategies. In the future, the use of genetically engin-eered cells for transplantation purposes also seems tobe a realistic possibility."9 However, it must beconcluded that at present cell transplantation into thehuman brain is at an early, experimental stage. Themechanisms underlying the reported improvementsafter transplantation in patients with Parkinson's

disease must be clarified in detail. Above all, it remainsto be demonstrated that grafted cells can survivepermanently in the human brain affected by variousdisease states. Obviously, for further progress in thisfield, it will be vitally important that basic animalstudies are carried out in parallel to well-designedhuman trials.

Our own research reviewed in this article was suppor-ted by the Swedish MRC (14X-8666), RiksbankensJubileumsfond, Rut Peterssons Donationsfond andThorsten and Elsa Segerfalks Stiftelse. I thank AndersBj6rklund, Patrik Brundin, Siv Carlson and RonMandel for valuable help with the preparation of themanuscript.

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Transplantation into the human brain: present future possibilities - … · 6-OHDA lesioned rats.'34 Amphetamine-induced rotational asymmetry can be reversed by grafts re-innervating

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