STATEMENT OF THE PROBLEM
The belief that the Central Nervous System (CNS) is capable of little or no regeneration or repair after injury was developed more than a century ago when experimental and descriptive neuroanatomy was gaining recognition as an important scientific discipline. At that time, the emphasis was on describing the morphology of different types of neurons in different brain regions and in tracing the connections among them. In fact, Santiago Ramon y Cajal (Portero-Sanchez, 1987), who performed some of the most detailed and extensive descriptive research ever done on the anatomy of the brain, became convinced that the regenerative capacity of neurons is lost by the time organisms reach maturity and that when the CNS is injured, little or no regeneration or repair is possible. Although the methods and techniques have become much more sophisticated and molecular, the paradigm underlying contemporary neuroanatomical research has tended to remain centered on anatomical connections, leading to pessimism about the potential for significant functional remediation and rehabilitation after CNS damage.
Fortunately, although much of the work is still in the experimental and pre-clinical stages, new developments in both pharmacological and surgical treatments for CNS trauma have led biomedical researchers to challenge Cajal's dogma and to begin thinking that recovery of function in brain-damaged adult patients may be attainable. Although there are many new and exciting ap- proaches to the treatment of brain and spinal cord injury, one that has captured the most public and professional attention has been neural transplantation, an area of neuroscience that has developed into its own specialization.
There has been great interest in considering transplants as treatment for degenerative disorders such as Parkinson's disease (PD), Huntington's chorea and Alzheimer's Disease (AD), (for reviews, see Goetz et al., 1993; Cassel et al., 1992a; Freed, 1991) In practice, attempts have been made to graft human fetal tissue directly into the brain of seriously impaired patients, although such attempts have been met with criticism on both methodological and moral grounds (Greeley et al., 1989; Neuhaus, 1989; Lindvall et al., 1990). Lindvall and colleagues (1990) used spontaneously aborted fetal tissue in one patient with Parkinson's disease and reported what appeared to be modest improvements. Madrazo and his colleagues (1988; 1990) have also reported success with such grafts in patients with PD, but their work has also been questioned even by those who have been pioneers in neural grafting research (Freed et al., 1990), because it has been difficult to replicate the beneficial effects due to inclusion of some subjective scales of measured improvement and undefined transient complications. The results of similar work by Lopez-Lozano, et al. (1990) with im- planted pieces of autologous adrenal medulla that had been perfused with enriched culture medium also included subjective evaluations, and all 20 PD patients remained on L-dopa medication throughout the course of the evaluation. By 12 months after the implant "there appeared to be slight overall deterioration, not yet statistically significant."
A number of recent studies have dealt with implants of human fetal mesencephalic cells to the basal ganglia of PD patients and patients with MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)- induced parkinsonism (Redmond et al., 1993; Spencer et al., 1992; Freed et al., 1992; Widner et al., 1992; Hoffer et al., 1992), three of which are reviewed in a recent article by Gage (Gage, 1993). The reports are difficult to compare to each other, due to methodological differences in the surgery, such as in fetal tissue age and processing prior to implanting, number of fetuses used in each implant, unilateral or bilateral implantation, and also to differences in drug treatment after surgery.
Each of the surgical teams reported some improvement in motor performance in some patients, and their evaluations had in common the use of several specific standardized rating scales, where the patient was rated compared with his own pre-surgery performance. The Spencer et al. (1992) study also included a control group of PD patients who did not receive transplants. In this study, although there appeared to be a trend towards graft-improved performance in the Schwab-England Activities of Daily Living (ADL) scores, the patients with implants did not differ significantly from the control group. The Redmond et al. study (1993), using comparable standardized ratings, reported significant differences on neuro- logical tests between control patients and patients with trans- plants.
Importantly, in the studies above, follow up studies were reported for up to 4 years post-implant, which allowed for comment on "the lack of serious physical or mental side effects" (Spencer, 1992). However, only the Redmond study reported on results of any specific cognitive, memory, or psychological tests other than those related specifically to PD testing. Use of more varied cognitive testing, not necessarily specific to PD motor symptoms, would give firmer support to assessments of long-range effects, some of which may be unexpected. At present, in considering the improvements cited, it is also important to consider the point made by Hoffer and coworkers, who state that, although "a small but long-term improvement" was shown by one of two of their PD patients receiving a fetal graft, "these improvements ... have not alleviated her basic neurological impairment." (Hoffer et al., 1992)
It is difficult, at this stage in transplant research, to evaluate properly the effect of the continuing pathology of the disease itself on the survival of grafted cells. Interestingly, the Widner study mentioned above deals with MPTP-induced parkinsonism. Whether the acute drug-related onset may be associated with a different prognosis for graft survival is unknown. The MPTP model has provided for advances in understanding the etiology of PD and for testing therapeutic measures. However, there may be differences between the progressive nature of PD and physiological events subsequent to the drug-related acute onset of MPTP induced parkinsonism that will need to be considered in evaluation of animal research using the MPTP model.
Given the current lack of supportive autopsy findings on the survival of fetal grafts, taken together with the generally positive reports with recent PD transplant patients , it is vital to establish, in animal models, the long-range viability of transplants used in PD treatment. Reports of survival of adrenal medullary grafts have yielded disappointing results, and autopsies have shown minimal or no survival of these autografts (Forno and Langston, 1991; Naini et al., 1991; Kordower et al., 1991; Hirsch et al., 1990; Waters et al., 1990; Jankovic et al., 1989; Peterson et al., 1989; Hurtig et al., 1989). Therefore, beneficial effects from grafting may be through mechanisms other than connections with the host or from prolonged release of dopamine by the graft.
Several recent studies have examined l-dopa activity after transplant. Positron emission tomography (PET) studies with ligands based on l-dopa have shown increased uptake in the operated putamen up to 18 months post-transplant (Hoffer et al., 1992; Sawle et al., 1992). It is not known, however, whether the increase noted can be correlated with graft survival, or whether it represents other transplant procedure-induced alterations in brain chemistry. The mechanism by which a graft may exert long-term effects needs to be determined. Without knowledge of the mechanism of action, it is difficult to say whether grafting techniques need to be refined or whether less intrusive methods may be as effective.
In the clinical setting, therefore, there are problems which have made replication of results difficult. First, the criteria for measuring and evaluating what constitutes functional recovery has varied from one group to another. This inconsistency might be alleviated by more standardized testing as was seen in the studies mentioned above. Second, careful controls for the effects of the grafts and/or surgery itself must be included in human studies.
With respect to basic research findings relevant to the transplant issue, extensive primate studies have not yet been done. This means that patients are being subjected to procedures for which the long-range prognosis is unknown. If the recent revelations on damaging long-term effects of breast implants contain a message, it is that we must examine the long-term effects of intrusive procedures before considering routine use.
No significant reduction in dose of L-dopa therapy accompanies transplant therapy. In one interesting report, Steece-Collier, et al. (1990) found that twice-daily injections of Sinemet (a combination of L-dopa and carbidopa, which inhibits the decarbox- ylation of peripheral L-dopa) to rats over a 6-week period blocked the development of grafted fetal dopaminergic cells. There was a marked reduction of tyrosine hydroxylase immunoreactivity and a decline of neurite outgrowth as well as increased infiltration of macrophages suggestive of an immune rejection reaction. Chronic administration of L-dopa has been shown to impair the morphological development of grafted embryonic dopamine neurons in vivo and to impair the survival of such fetal dopamine neurons in culture (Steece-Collier et al., 1990). In a study by Madrazo et al. using grafts of adrenal medullary tissue , optimum results were obtained in a patient who was unable to tolerate l-dopa therapy (Madrazo et al., 1987). To date, most PD transplant patients must continue to receive L-dopa therapy for symptom control; although this treatment could result in the eventual destruction of the graft as well as inhibition of its functions and long-term effectiveness.
The issue of when and how to use human embryonic brain tissue grafts, grafts of autologous tissue, or even grafts of genetically engineered cell lines is being actively discussed and debated by both clinicians and basic researchers alike. Very few controlled primate studies have been conducted. Many neuroscience research- ers, including the present authors, are deeply concerned that there are not yet sufficient data to warrant routine application of embryonic tissue grafts to the treatment of traumatic or degenera- tive neurological disorders. Because the literature on this topic is now so large, we will focus selectively on two issues relevant to the use of neural transplants in the treatment of brain and spinal cord injuries. The first issue concerns possible mechanisms through which transplants may act to facilitate recovery and whether similar beneficial results may be obtained with less intrusive methods. Initial thinking held that embryonic brain tissue grafts would form connections to the host brain and thus could be used to "replace" neurons that were lost or damaged as a result of trauma or degenerative diseases of the nervous system. However, as we learn more about the various processes underlying functional recovery from brain damage, the "connectionist perspec- tive" is gradually shifting to encompass other potential mechanis- ms, such as neurotransmitter replacement or trophic support, by which grafts can serve as therapy for CNS trauma and degenerative disorders (Labbe et al., 1983; Stein et al., 1985). Each of these mechanisms of transplant action has been identified in the frame of specific lesion paradigms using animal models. While it is theoretically possible that there may be some overlap in mode of action of one specific transplant in one specific paradigm, the focus of such research is to elucidate the specific mechanism under investigation. The second issue concerns possible problems in transplant treatment, such as genesis of epileptic seizures, immunological rejection, and, in cases of fetal transplant, the need for consideration of health status of fetus and mother and the effect of gender differences in both organ donor and patient re- sponse.
TRANSPLANT-MEDIATED RECOVERY
BY RESTORATION OF NEURONAL CIRCUITRY
Despite the large body of evidence supporting the argument that fetal grafts can restore neural and behavioral functions without the specific reconstruction of neural circuitry, the notion still remains that "re-wiring" of pathways could be the most effec- tive means to enhance recovery.
While many investigators have observed that there are recip- rocal connections between the transplant and host brain tissue, close inspection usually indicates that such connections are sparse and limited to the surface contact areas between the graft and host tissue. In addition, both suspensions of fetal neurons as well as 'solid' grafts, have individually healthy neurons, but the overall cytoarchitecture of the graft is not normal (Stein & Mufson, 1987; Mufson et al., 1986). Nonetheless, there are numerous anatomical studies (but rarely with behavioral follow-ups) demonstrating some type of point-to-point reestablishment of circuitry and synaptic morphology. In a few of these cases, the restoration of connections may be important for the initiation of physiologic and functional recovery.
Sotelo and Alvarado-Mallart (Sotelo and Alvarado-Mallart, 1987a; 1987b; 1991) performed grafting experiments using the mutant mouse pcd (Purkinje-cell degeneration), with grafts of embryonic cerebellar primordium taken from isogeneic C57BL mouse embryos. They report a highly specific migratory invasion into the deprived cerebellar cortex by the grafted Purkinje cells. These grafted cells proceed through normal developmental processes, forming dendritic arbors and integrating synaptically with sprouting host axons. However, not all sprouting axons are able to contact the newly established Purkinje cells, and frequent "hypertrophic aberrant plexuses" are noted. Sotelo and Alvarado-Mallart also conclude that "the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit." (Sotelo and Alvarado-Mallart, 1991)
Mudrick, Bainbridge and Peet (1989) used an ischemic model of hippocampal damage to examine reestablishment of circuitry and to describe the electrophysiological responsiveness of transplanted hippocampal neurons. One week after damage, the hippocampus was repopulated by suspensions of E-18 hippocampal neurons injected into the dorsal hippocampus. Single-unit recordings were made in vitro from hippocampal slice preparations 2-9 months after the initial grafting procedure. In most of the cells recorded, general membrane properties were "similar to those reported for normal CA1 pyramidal cells and were not obviously different from control pyramidal neurons in the same bath" (Mudrick et al., 1989). In addition, synaptic potentials in transplanted neurons could be evoked by stimulation of cells in the host brain. However, IPSPs that normally follow EPSPs in pyramidal cells were not observed. Thus, inhibitory mechanisms were not present in the transplanted neurons -- despite the fact that the "transplanted neurons were morphologically similar to hippocampal pyramidal neurons (p.338); that is, apical and basal dendritic arborizations formed in a normal orientation.
Several studies have addressed the survival of homo- vs. heterotopic grafts (Heuschling et al., 1988; Clarke, Nilsson, Brundin and Bjorklund, 1990). Clarke and coworkers examined the synaptic connections formed by grafts of cholinergic neurons into the damaged hippocampus of adult rats. The authors used fetal tissue taken from a variety of cholinergic areas and transplanted the tissue unilaterally into the hippocampal formation of rats with ipsilateral transection of the fimbria-fornix. This was done in order to determine if cholinergic tissue of different origins would have the same potential for forming synaptic connections when grafted into the hippocampus. The rats were allowed to survive 14- 17 weeks before their brains were taken for light and electron- microscopic evaluation of cholinergic innervation using acetyl- cholinesterase (AChE) and choline acetyltransferase (ChAT) cyto- chemistry. Although the same number of cells were injected, only the septal grafts were "able to reinnervate the host hippocampus completely and in all subfields," despite the fact that the spinal cord and nucleus basalis magnocellularis (NBM) grafts were the largest. Also, the septal grafts had the largest number of ChAT- positive cells and showed "normal or near normal distribution and the same morphological characteristics as the normal septo- hippocampal cholinergic fibers". At the EM level, septal cells had the largest number of ChAT-positive boutons. Based on their observations, Clarke, et al. (1990) concluded that "only cholinergic neurons obtained from the septal-diagonal band areas achieved a reasonable degree of reinnervation and reestablished a near normal pattern of synaptic activity."
We need to consider first that these studies were performed in rodents, not humans, and the behavioral consequences of the "re- stored circuitry" have not been evaluated. Second, the authors intimate the reestablishment of specific neuronal pathways, but at the same time they are careful to make statements that the connection are "similar to normal counterparts" (Mudrick et al., 1989) or "fairly normal" (Clarke et al., 1990). It is important to note also, that in the Mudrick, et al. experiment (1989), the transplanted hippocampal neurons had electrophysiological charac- teristics similar to normal cells except for the fact that the neurons did not exhibit any inhibitory post-synaptic potentials (IPSPs). This 'minor' difference between grafted and normal hippocampal tissue was what led Buzsaki, et al. (1989) to suggest that such transplants, when integrated into the host brain, serve as epileptic foci. Third, one has to be careful in selecting the specific tissue to be grafted -- as Clarke, et al. indicated (1990), not all cholinergic tissue does well in making appropriate connections with the host hippocampus; biochemical conditions at the transplant site apparently also need to be considered when selecting embryonic tissue for grafting.
Finally, we need to consider that even partially aberrant "rewiring" could have dramatic, long-term consequences for func- tional recovery and rehabilitation (Buzsaki et al., 1989). For example, neural tissue grafts that form even limited connections with the host brain may lead to both physiologic and behavioral seizure induction either spontaneously or under some conditions of environmental excitation (e.g. stress, drug interactions, other diseases, etc). In the absence of precise long-term behavioral assessment in both human and animals, it is not possible to assess possible deleterious effects of abnormal connections accurately.
REPLACEMENT OF NEUROTRANSMITTERS BY NEURAL TRANSPLANTS
When a brain region is injured or when there is significant degeneration as in PD, there is often a loss of neurotransmitters necessary to mediate normal functions. Although L-dopa therapy has been partially successful in the treatment of this disorder, there are a number of problems associated with its chronic application, including resistance to the drug and the "on-off" phenomenon which makes life difficult for the patient. Additionally, the efficacy of L-dopa therapy depends on the remaining population of nigrostriatal dopaminergic neurons. Interest in tissue grafts for PD patients arose because it was thought that "internalized" therapy would provide for a steady and sustained release of dopam- ine and/or its precursors directly into the brain. Work by Perlow, et al., (1979) and Freed et al. (1981) using rats as subjects, and subsequent studies with non human primates (Sladek et al., 1988; Hansen et al., 1990; Taylor et al., 1990), suggested that either dopaminergic fetal brain tissue or autologous grafts of adrenal medulla, transplanted in or near the damaged striatum, could reduce some of the behavioral deficits that mimic PD symptoms seen in human patients. It was this thinking that initially led to the placement of autologous adrenal medullary grafts into the brains of PD patients (Backlund et al., 1985). Although limited to moderate success has been reported, the relative value of long-term clinical benefits has been hotly contested and debated (Stein, 1985; Lindvall et al., 1989; Yong et al., 1989; Freed et al., 1990; Ahlskos et al., 1990; Lopez-Lozano et al., 1990).
At the level of basic research, the fact that grafts of adrenal medulla suggested some degree of functional benefit, although neurite outgrowth was unlikely, indicated that neuro- transmitter replacement or other factors secreted or stimulated by grafts could be as important as the formation of new connections in mediating recovery from brain damage. This latter view received support from the recent study of Bankiewicz, et al. (1991) who examined the effect of grafting fetal cerebellum or spinal cord into rhesus monkeys with MPTP-induced parkinsonism. The authors observed that the implantation of nondopaminergic tissue resulted in significant symptom reduction within 2 months after surgery; the improved behavior remained stable over the next 6 months. The grafts were shown to induce neuronal sprouting of dopaminergic fibers from areas of the host brain that were not affected by the MPTP. Bankiewicz, et al. (1991) suggested that both nondopaminer- gic grafts and reactive astrocytes (responding to the inflammation after surgical trauma) could secrete trophic factors that stimulate neuronal sprouting in the host brain, resulting in an increase of dopamine and behavioral recovery.
Consideration is also being given to neurotransmitter replacement therapy in disorders associated with cholinergic innervation, which occurs in AD, traumatic injury, ischemia or stroke. Recently, Ikegami, et al. (1989) used a potent neurotoxin (AF64A) injected into the lateral ventricles of rats in order to kill cholinergic neurons of the basal forebrain which project to the hippocampus and neocortex. When this drug is given there is a significant decline in the activity of the cholinergic markers, AChE and ChAT. This loss of cholinergic activity is associated with impairments in learning and memory that some think may mimic the deficits seen in senile dementia of the Alzheimer's type (SDAT) (Bartus et al., 1982; Coyle et al., 1983; Gage et al., 1984). One week after the neurotoxin was administered, Ikegami and colleagues injected a dissociated suspension of 17 day-old fetal septal cells (primarily cholinergic) directly into the dorsal hippocampus. Three months after this surgery, the rats were killed and their brains examined for ChAT and AChE activity in the hippocampus, neocortex and striatum.
In comparison to normal controls, animals treated just with AF64A showed an almost complete loss of AChE staining in the hip- pocampus, indicative of long-term hypoactivity of cholinergic neurons projecting to this structure. In contrast, the septal grafts produced a dramatic increase in AChE fibers projecting from the transplant into the host brain, and there was also a sig- nificant recovery of ChAT activity on the grafted side of the brain. ChAT levels on the non-grafted side were reduced to 38-46% of normal, depending on the region assayed; whereas ChAT recovered to an average of 67% of normal on the grafted side and some rats showed 78% of normal levels following grafting.
More recently, Nilsson, et al. (1990) placed either solid blocks or dissociated suspensions of 15-16 day-old embryonic septal tissue into the right hippocampus of adult rats with unilateral lesions of the fimbria-fornix, which were created to eliminate septo-hippocampal cholinergic fibers. Nilsson, et al. showed that while lesions caused a 70% drop in acetylcholine (ACh) levels, compared to normal controls, either suspensions or solid grafts of embryonic septal tissue could augment ACh levels in rats with fimbria-fornix lesions. Indeed, the grafts of septal tissue restored ACh levels to significantly higher than normal, indicating an "overshoot" response, which could be considered an aberrant response caused by the transplants.
Several other studies suggest that grafts providing neuro- transmitter replacement (instead of point-to-point neuronal recon- nections) can potentiate behavioral recovery, but the effects are not without certain complications that would need careful attention in considering such an approach for the treatment of human disorde- rs. For example, Segal et al. (1989) found that embryonic septal grafts in rats with fornix transections did not facilitate behavioral recovery on a water maze spatial navigation test for reference memory, given 5 months post-surgery. Subsequent AChE histochemistry revealed that the graft did extend AChE-containing fibers into the host hippocampus. However, when the rats were given injections of physostigmine, an inhibitor of AChE, those with grafts showed significant improvement compared to rats with lesions alone. ACh produced by the septal grafts was not sufficient to ameliorate the lesion-induced deficits. Only with the addition of physostigmine was there improved performance. Thus, although the transplants grew in the host brain and appeared to form connections in the host hippocampus, behavioral recovery was very limited and could not occur without additional pharmacological manipulation. The necessity for additional pharmacological treatment is similar to findings in humans with PD receiving grafts of fetal tissue or of autologous adrenal medulla. The grafts are thought to function by providing dopamine, formed from catecholaminergic precursors endogenous to the graft, in a curtailment of the normal synthetic pathway to norepinephrine and epinephrine. However, continued treatment with L-dopa is required for reduction and control of symptoms.
This difference is addressed in the work of Hodges et al. (1990), who have examined the ability of cholinergic grafts to facilitate recovery after damage to the septum and the NBM (nucleus basalis magnocellularis) in adult rats. These lesions allowed exploration of the removal of cholinergic input to the hippocampus and neocortex, respectively. Hodges and coworkers then injected E- 15 day old cholinergic-rich basal forebrain grafts into the dorsal hippocampus and/or the neocortex (the target areas of cholinergic projections), or into NBM (the area of origin of the damaged fibers). The rats were given two weeks of post-operative rest and then tested for 14 weeks on a radial arm maze task to examine the time course of the functional recovery. Their results demonstrated that cholinergic transplants into the hippocampus (or into the neocortex) "fully restore radial maze working memory deficits in brain damaged animals", and to some extent improved reference memory as well. According to the authors, transplant growth was very vigorous and, as a result, the hippocampal architecture was very distorted. Consequently, it was thought unlikely that the recovery could be due to neuronal "rewiring" since there was so much hippocampal distortion, and similarly surviving grafts into the damaged nucleus basalis were not effective in enhancing be- havioral recovery.
Interestingly, the results of Hodges et al. are similar to those of Segal et al. (1989) in that transplants made the rats more sensitive than intact animals to both cholinergic agonists and ant- agonists often used in memory research (scopolamine, mecamylamine, nicotine). In fact, injections of nicotine significantly impaired performance in rats with cholinergic transplants, while learning was faciliated in rats with cholinergic lesions by dose levels of nicotine which produced no learning effects on normal rats. Thus, while some neuronal grafts may serve to potentiate recovery from brain injury, drug interactions may have to be carefully monitored since the transplants may produce a "high dose effect" or a form of long-lasting "supersensitivity" to certain pharmacologic agents. Recently, Will and colleagues (1989) have reported that some types of grafts produce 140-240% above-normal increases in neurotransmit- ter levels or their precursors and that, under such conditions, the transplants could actually lead to more deleterious effects on behavior than lesions alone, a result that the authors suggest may be the result of graft-induced neuronal toxicity caused by the overproduction of excitatory amino acids. In considering possible clinical application of transplants one should understand that excessive levels of neurotransmitters or their precursors could be dangerous to the patient. Heightened neurotransmitter activity could, for example, cause further loss of neurons over time or, in lesser amounts, perhaps cause paroxsymal activity or seizures, especially if other drugs, such as tranquilizers, beta-adrenergic blockers, steroids etc, are given concomitantly.
With respect to the appropriateness of transplant "therapy" for cognitive disorders, we must also be concerned with the question of what specific type of tissue is most effective in promoting the hoped-for functional recovery. Some workers have noted that, in diseases affecting memory such as senile dementia- Alzheimer's type (SDAT), other neuroregulatory systems such as noradrenergic neurons from the locus coeruleus and serotonergic neurons from the raphe are also implicated in addition to choliner- gic loss (Decker and McGaugh, 1991). For example, senescent rats show significant declines in both catecholamines and cholinergic titres as well as deficits in spatial learning and memory (Collier et al., 1985; 1988). Providing replacement therapy for only one neurotransmitter could actually lead to an 'imbalance' resulting in more abnormal, rather than improved function(s).
To examine this question in more detail, Nilsson and col- leagues (1990) created lesions that damaged both the serotonergic (by 5,7-DHT lesion) and cholinergic forebrain (by radiofrequency lesion) projection systems to the hippocampus. Ten months after surgery, animals receiving combined raphe and septal grafts were no longer impaired on a water maze task in comparison to normal animals. In contrast, all the other groups having individual grafts of either septum or raphe continued to perform poorly, although the individual grafts were shown histochemically to produce substantial reinnervation. This was in contrast to this group's earlier work using a different lesion technique (Nilsson et al., 1987), where following fimbria-fornix transection, single intrahippocampal or intracavitary septal grafts were sufficient to produce significant attentation of memory deficits assessed in the Morris water maze.
The studies above suggest that restitution and balance of multiple neurotransmitter systems in CNS disorders must be taken into consideration in thinking about application to the clinical situation. In some disease states, restitution of more than one neurotransmitter may be found to be more useful in effecting maximum recovery.
TROPHIC FACTORS, BEHAVIORAL RECOVERY AND NEURAL TRANSPLANTS
As we have seen from the section above, neural transplants can have functional consequences attributable to the stimulation and/or release of neurotransmitters rather than by the restoration of damaged circuitry per se. In addition, neural tissue grafts are known to make factors which, at least in vitro, promote cell survival, enhance neurite outgrowth and stimulate the differentia- tion of both peripheral and CNS neurons (Cotman & Kesslak, 1988). Brain injury itself stimulates the production of trophic factors (Nieto-Sampedro, 1988) which help damaged neurons to survive the secondary consequences of traumatic injury by blocking the effects of toxic factors and promoting synaptic regeneration and repair. In fact, Nieto-Sampedro, et al. (1984) demonstrated that brain injury "wound extract" facilitated the survival of some grafts. Thus, inserting graft tissue into the damaged neocortex, immediately after injury, led to less survival than when the grafts were made 7-10 days after the initial lesions, when the titres of survival-promoting neural factors were at their peak.
Several studies have examined the issue of whether neural grafts mediate functional recovery by the stimulation or release of trophic or survival factors. The issue is important for several reasons. On the one hand, if some grafts mediate functional re- covery by production or release of trophic factors, the beneficial effects could persist even if the transplants were removed or degenerated at a later time. On the other hand, if specific neuro- transmitters or specific circuits are essential for the return of function, then the permanent presence of the transplant itself would be necessary to maintain the recovery process. If functional recovery can be induced and sustained by trophic factors alone, it might then be possible to inject the substances systemically or directly into CNS and thus avoid the additional risk to the patient of further brain surgery and its attendant complications, costs and risks.
One of the first series of studies to propose the "trophic hypothesis" of transplant-induced recovery came from our laborator- ies (Labbe et al., 1983; Stein, 1988). In the initial experiment, adult rats were given bilateral lesions of the frontal cortex and E-19 solid grafts of frontal tissue seven days later. When tested on a delayed spatial alternation T-maze task (DSA) five days postoperatively, the transplants led to significantly better post- operative acquisition of the spatial learning problem than was seen in rats with frontal lesions alone, although the animals with transplants did not perform as well as intact rats. The functional recovery we observed was occurring too rapidly to be explained by the formation of new circuitry to replace that lost by the injury.
Nieto-Sampedro (1983) and Cotman (1984) and coworkers showed that neurotrophic activity in adult rat brain supporting transplant growth declines after peaking at approximately 15 days, at which time scar tissue also could form, blocking transplant-host intera- ctions. There may then be a critical "window-of-opportunity" during which grafts may optimally be placed into the damaged brain.
We examined this notion (Stein & Mufson, 1987) using groups of rats with bilateral frontal cortex injuries who then received solid frontal grafts at 7, 14, 30 or 60 days after the initial surgeries. All groups began testing on the DSA task beginning 7 days after the grafts were introduced. Our results provided us with some useful and practical information and helped to strengthen the "trophic hypothesis" of functional recovery.
First, although the 7 day and 14 day transplant group showed significant functional recovery, it was very clear that neither the 30 or 60 day delay in graft treatment was effective in reducing learning impairments on the spatial task. Subsequent histological follow-up revealed that there was very little graft survival in any of the rats in the 14, 30 or 60-day transplant groups, although grafts in the 7-day group grew successfully to about eight times their initial size and had relatively large connecting bridges with the host brain tissue.
In light of the improvement in learning behavior in the 14-day transplant group, where there was little graft survival, it would be difficult to argue that the sustained behavioral recovery seen was due to the formation or re-establishment of reciprocal connections between the host and transplanted tissue. Moreover, it would also be unlikely that recovery was the result of continuing neurotransmitter replacement emanating from the grafts because the functional benefits were maintained in the 14-day group even though the grafts themselves did not survive; thus sustained release of transmitters by the grafts could not be assured.
Thus, at least for frontal cortex injury, waiting too long after the initial damage to introduce grafts may result in very poor survivability and, importantly, in no functional recovery. The "window of opportunity" for successful grafting (or other forms of treatment) may vary according to the type of initial injury as well as which parts of the nervous system are damaged. Although little research has been conducted specifically addressing the age of the patient and his/her health status, these factors may also play an influential role in extending or shortening the "window" available for brain injury repair.
Although the above studies provide good evidence for the validity of the trophic hypothesis of transplant-induced recovery in this system, another issue remained. If transplanted tissue were removed after producing functional recovery, and deficits reappeared, the presence of the transplant for either transmitter replacement or re-establishment of circuitry would be a more likely mechanism of recovery. If, on the other hand, transplants were removed after recovery has occurred and the recovery was sustained, it would be very hard to argue strongly for the connectionist view and it would also make the need for continuous transmitter replace- ment as a condition necessary for recovery much less forceful.
Several studies have examined the issue of transplant removal and persistence of recovery. In our laboratory (Stein, 1988), we found that rats with E19 frontal cortex transplants showed significant recovery in DSA as compared to lesion-only controls. After this testing, the transplants were removed from one group of rats, and all animals were tested 3 months later on a spatial navigation test along with retesting for DSA. The rats with transplants removed performed as well on the acquisition of spatial navigation learning as those with transplants intact. Both groups performed better than the lesion alone controls, but they were not as good as the intact animals. Both the transplant-removed and the transplant-intact groups retained the original DSA learning and were significantly better than the lesion alone group. Removal of the grafts once recovery had occurred did not re-establish the deficits caused by the original frontal cortex injuries. Destruc- tion or total removal of the grafts after a relatively long period in the host brain would certainly eliminate most reciprocal connec- tions and any continuing transmitter 'replacement' they provided. At least with respect to frontal cortex injury then, the graft removal study demonstrates that the integrity of the graft is not a necessary condition for the maintenance of functional recovery although it may be important for initial reorganization of function.
Woodruff, Baisden and Nonneman (1990) reexamined this issue using the hippocampal lesion model to determine if the presence of grafted fetal hippocampal tissue was necessary to maintain the functional recovery. Woodruff et al.'s findings were essentially the same as ours. Rats with transplants and hippocampal lesions did better on a bar press withholding response (DRL-20) than lesion alone animals but not as well as intact controls. Removal of the transplants once the recovery had occurred did not lead to further deterioration of performance. Woodruff et al. suggested that the transplant circuitry was not necessary for continuing behavioral recovery, and speculated that cells of the transplant, in particular glial cells, might act to remove excitotoxins, thus stabilizing the environment. Although glial cell grafts may help to promote recovery by secreting trophic factors directly or by stimulating the release of trophic survival factors by the host brain tissue, they may also protect vulnerable neurons by removing injury-induced excitatory amino acids that are toxic to neurons. Such findings are particularly relevant to injury models of damaged hippocampus, due to the high sensitivity of this structure to glutamate toxicity (Mattson & Ryuchlik, 1990).
Kesslak et al. (1986) have provided evidence that either occipital wound extract from neonatal rats or transplants of reactive astrocytes from neonatal rats facilitated recovery in acquisition of DSA in adult rats with bilateral frontal cortex injuries. The trophic hypothesis may then be taken to suggest that functional recovery from traumatic brain injury or degenerative disorders of the CNS may be due to non-neuronal factors that can be directly manipulated. The data on glial cell grafts, taken together with the transplant removal studies, strongly suggest that there are reasonable, pharmacological alternatives to the use of fetal brain tissue grafting in treating brain damage or degen- erative CNS disorders. One such alternative to neural grafting might be the direct, intracerebral administration of Nerve Growth Factor (NGF) to facilitate morphological and behavioral recovery from damage to the forebrain cholinergic system (Hart et al., 1978; Will et al., 1988). While use of NGF might involve direct infusion to the brain, treatment with one (or several) specific pharmaco- logical agents would provide a much more controlled setting. Some trophic factors, such as gangliosides, can be administered parente- rally and are proving useful in treating a variety of CNS degener- ative disorders as well as traumatic injuries (Dunbar & Stein, 1988; Mahadik & Karpiak, 1988). The experiment by Kesslak also raises the issue of homology of tissue. In another study from our laboratory (Stein et al., 1985), we asked whether transplants could reduce some of the visual deficits caused by bilateral lesions of the occipital cortex. There was a 7 day delay between initial injury and grafting and then only a short delay between introduc- tion of the grafts and behavioral testing for brightness and pattern discrimination learning. In this experiment we used two types of fetal tissue taken at embryonic day 19 (heterologous frontal cortex and homologous occipital cortex) in two separate transplant groups. We observed significant functional recovery on brightness, but not pattern discrimination, in rats given frontal cortex grafts into the damaged occipital cortex. Surprisingly, despite the fact that embryonic occipital cortex grafts were as large and as viable as the frontal cortex transplants, this tissue did not result in any functional recovery. After approximately 4 months survival, vigorous transplant growth was seen, but tracing techniques showed no evidence that any occipital or frontal-derived grafts were forming reciprocal connections with the host CNS. We concluded that the behavioral recovery seen was not due to the rebuilding of neuronal circuitry. We were also forced to conclude that homologous tissue may not always be the most effective in promoting functional recovery.
Evidence from Przedborski et al. (1991) suggests that grafting of fetal liver tissue was as effective as implants of fetal striatal tissue in protecting rats with striatal lesion from 6- OHDA-induced rotational impairment and decrease in dopamine uptake sites. Additionally, sham transplantation resulted in protection from behavioral impairment and biochemical alteration. These types of experimental controls cannot be morally justified in the treatment of patients, but nevertheless, Prezedborski's et al.'s findings reinforce the notion that neural grafts may not elicit improved recovery through "replacement" mechanisms per se. The results also raise the issue of whether heterologous grafts and/or the surgical procedures alone provide the impetus for alterations in the host brain milieu which result in improved behavior and a more "normal" chemical environment.
In a recent commentary on transplants in PD patients, Landau (1990) provided a summary of the considerable body of literature generated concerning neurosurgical, non-transplant treatment. Prior to introduction of l-dopa therapy, cerebral hemisphere lesions in PD patients were explored, with reports of rapid improvement, especially in attenuation of involuntary movement (Cooper, 1956; Cooper, 1961; Cooper, 1960). In some cases, improvement of PD symptoms was reasonably prolonged. More recently, Bergman et al. (1990) demonstrated that lesions of the subthalamic nucleus in monkeys rendered parkinsonian by injection of MPTP into the substntia nigra, eliminated all of the motor symptoms associated with the disorder. Here, the creation of additional damage produced an almost immediate remission of akinesia, rigidity and tremor. This finding is similar to the report of the beneficial motor results of subthalamic hematoma in a PD patient (Sellal et al., 1992). While an alternate invasive surgical approach for PD patients may not be the final answer to appropriate treatment, much can be learned from animal studies using this model, in which recovery is induced without recourse to the need for fetal tissue grafting.
The point then becomes, as stated by Landau, "not whether fetal or mature, adrenal or brain tissue can be successfully transplanted into mammalian brain" [but] "whether the symptoms of experimental parkinsonism can be specifically remedied by the physiologic activity of the transplanted tissue." (Landau, 1990) It may be that the symptoms can be remedied equally well by other, less risky surgical manipulations.
Much clinical work has focused on imposition of tissue that is homologous with respect to tissue type or to function (e.g. adrenal tissue for precursors of dopamine). However, results of research such as were generated by the liver transplant experiment demonstrates that there are many unknown factors involved in transplant success. When we find why tissue that is heterologous can, in some models, provide demonstrably better recovery of function than homologous tissue, we may be closer to understanding trophic actions of transplants. Such knowledge would allow for development of more refined therapies, including application of highly specific agents promoting survival or regeneration. In this context, the advantages of using autologous donor tissue in PD transplant work has been questioned, following reports of decreased catecholaminergic content in parkinsonian adrenals (Carmichael et al., 1988). The patients adrenals also contained cytoplasmic inclusion bodies thought to be related to metabolic abnormalities in PD, which result in Lewy bodies in the substantia nigra (den Hartog Jager, 1970).
NGF has been reported to increase the survival and outgrowth of adrenal medullary cells in culture (Silani et al., 1990). Additional pre-clinical animal research may ultimately yield methods for promoting survival of grafts, perhaps through manipulations such as provision of additional nerve growth factor (NGF), as has been reported in animal models (Doering, 1992; Kordower et al., 1990) and in a human PD patient with adrenal medullary graft (Olson et al., 1991).
IMMUNOLOGICAL PROBLEMS IN NEURAL GRAFTING
As with any type of transplant surgery, one of the most important concerns is whether tissue rejection can be prevented. In the initial excitement over neural grafting and its potential for functional recovery, the question of immunological rejection was not given much attention. Partly, this was because of the long-standing belief that the brain is an "immunologically privileged site." Recent reconsideration of this problem, however, has given rise to the idea that the degree of immunological privilege as not absolute (for reviews of this problem, see Widner & Brundin, 1988; Sloan et al., 1991).
In human patients, there have already been several clinical reports describing "successful" grafting of human fetal brain tissue for the treatment of Parkinson's disease in which follow-up has been monitored for period greater than one year after the ini- tial surgery. To date, no long-term studies of immunorejection in humans have been presented and the data from animal studies that address this question are highly variable.
With respect to animal studies (where very often highly inbred strains of animals are used both as donors and recipients), it will come as no surprise that allografts seem to have a better chance of survival than xenografts; although even in the former case, results are far from unequivocal. In mice, for example, recent studies have demonstrated that with allografts of neural tissue, T-lym- phocytes are seen in the grafts within four weeks after transplan- tation (Nicolas et al., 1988). With longer time periods of 16-24 weeks post-transplantation, "there was a marked increase in mononuclear cell infiltrates and much greater areas of graft necrosis" (Nicolas et al., 1988).
Working with neonatal rats, Rao et al. (1988) found that neural grafts into neonates did result in "mild to moderate" lymphocytic infiltration "suggestive of an early spontaneous rejection reaction". The absence in the brain of classical antigen-presenting dendritic leucocytes and the presence of the blood-brain barrier (BBB) have been thought to contribute to pre- vention of a rapid immune response. However, it has been reported that activated lymphocytes are able to cross the BBB (Naparstek et al., 1984). The physical process of graft application itself can also compromise the BBB, providing antigenic access to lymphoid tissue and exacerbation of a central immune response. Rao, et al. (1988) make a very important point that should be carefully considered in the application of neural grafting to human patients. "While a clear picture of a single graft rejection mechanism has not emerged as yet, current experiments suggest a pattern of events. Essentially, the grafts exist in a metastable immune balance that can be disrupted by a variety of factors. Each one on its own may not be sufficient to provoke a rejection response, but once a critical threshold is reached, a cascade of events may occur which precipitates a classical immune response (p. 285)."
One example of a graft rejection-triggering mechanism could be the further disruption of the blood-brain-barrier (BBB) by a pathogen or by mechanical manipulation. Recently, Pollack and Lund (1990) implanted mouse fetal tissue into neonatal rats (a nonsynge- nic graft). One month after grafting, the rats were given an in- fusion of 1.4M mannitol into the internal carotid artery ipsilat- eral to the transplant. Within 3 weeks after infusion, histologi- cal examination of the brains revealed that the mannitol had com- promised the BBB and led to "a prominent expression of major histocompatibility complex antigens on cells adjacent to the graft". In turn, the BBB disruption reduced the immune protection of the foreign tissue grafted into the host brain rendering it susceptible to immune rejection, leading the authors to conclude that "...it is possible that circumstances such as viral infections that can lead to expression of foreign cell-surface antigens on brain cells may place these cells at risk in instances of barrier leakage" (p 119).
In an excellent review of factors influencing graft utility, Cassel and coworkers discuss several possible sources of rejection in both xenografts and allografts (Cassel et al., 1992a), including the antigenic properties of major histocompatibility complex antigens (Pollack et al., 1990; Hickey and Kimura, 1987), whose presence appears to precede graft rejection. Recent evidence also supports an immunoreactive role for host microglial cells (Poltorak and Freed, 1989). Cyclosporin A, an imunosuppressive drug used routinely in organ transplants, has been shown to enhance the survival of xenografts in animal models (see Widner and Brundin, 1988). As more becomes known about the limitations of immunological privilege in the brain, the probable necessity of administering immunosuppressant drugs along with transplants becomes apparent. Cyclosporin-A has also been used in a rat model of PD, where it was instrumental in allowing long-term survival of xenografts (Brundin et al., 1989). However, it was necessary to continue daily treatment with Cyclosporin-A for the entire time course of the experiment, an effect which is in keeping with the lifetime treatment necessary in organ transplants. Whether such extended treatment would be needed in allograft transplants remains to be determined. Cyclosporin-A itself is not without toxic side effects (Nozue et al., 1993; Hahn et al., 1992; Krueger et al., 1991). The population of patients severely affected by degenerative diseases is by its nature an older group. The effect of such a potentially toxic treatment on older patients could be a dramatic reduction of the patient's quality of life.
The problems highlighted by the various groups studying the immune reactions to neural grafts clearly indicate that many serious issues remain to be resolved before grafting becomes a routine procedure in the treatment of brain injury and disease. In the older patient with severe and potentially terminal disor- ders, considerations of slow immune rejection may not be as important -- if grafts would survive and function long enough to provide a better quality of life in the time remaining. For the younger patient, allografting treatments would be subject to a high risk of immunorejection and inflammatory reaction.
There is a body of literature suggesting that autologous, adrenal medullary grafts in humans can promote amelioration of Parkinsonian symptoms in some patients. However, there is less than complete agreement as to the clinical efficacy of this radical approach (Stoddard et al., 1989; Freed et al., 1990; Fine, 1990). Experimental evidence also exists in rodent and non-human primate models showing that transplants can sometimes result in significant recovery of function from different kinds of brain injuries.
As is the case for pharmacologic treatments designed to enhance recovery from brain damage (see Feeney & Sutton, 1987, Stein & Sabel, 1988 for detailed reviews), neural transplants will exert their effects through a variety of different processes and mechanisms, including neurotransmitter replacement, release of trophic substances, and possibly, in a few cases, through trans- plant/host neural connections.
Experimental models demonstrate, however, that we need to be aware of the possibility of aberrant connections, which may produce deleterious behavioral effects. We also need to explore the effects of grafts on "normal" plastic responses of the brain to specific types of injury, and the allied question of whether alteration of such "normal" responses is beneficial or not. For example, Cassel and coworkers (Cassel et al., 1992b) have shown that septal grafts into the dorsal hippocampus of female rats two weeks after lesion of the septohippocamp pathway result not only in increased cholinergic activity and increases in concentration of serotonin, but also in a reduction in noradrenaline concentration in the dorsal hippocampus, essentially a reversal of the effects of lesion alone. It was speculated that the decrease in NA could have resulted from either inhibition of sympathetic sprouting after cholinergic denervation or in reduction of NA emanating from sprouted fibers. Alteration of systems not specifically targeted by treatment may be a negative side-effect in treatments involving neurotransmitter replacement or trophic agent release. The treatment of PD is currently the primary clinical application of the transplant procedures. However, there is increasing awareness that PD is not only a dysfunction of dopaminergic neurons, but also affects other areas of the brain and other neurotransmitters. In attempting to apply transplant "replacement" therapy to a number of degenerative diseases, there is the real possibility of creating, instead, an imbalance in brain chemistry that could itself worsen, rather than ameliorate, the symptoms expressed.
Because of the problem of subjective assessment of ongoing human transplant procedures and the real possibility of deleterious effects of abnormal connections or clinically created imbalances, long-term, detailed behavioral follow-up studies should be done for any kind of transplantation treatment for brain injury. Within the last five years, hundreds of animal studies have been published in which the neuroanatomy and neurochemistry of the graft-host interactions have been examined. Only approximately 10% of the published papers report on the behavioral consequences of the grafts. For the most part, in animal models, embryonic brain tissue grafts have been successful in reducing (but not completely eliminating) sensory, motor and cognitive deficits caused by traumatic injury to the adult brain. However, results of the recent study by Woodruff et al. (1990) using a rat bilateral hippocampectomy model and transplant of embryonic hippocampal tissue, suggested that grafts may not always result in recovery, and may indeed make the animals worse on some tasks. Similar findings, with respect to behavioral impairment have also been reported with use of intrahippocampal fetal septal cell suspension transplants in female rats given selective fimbria-fornix lesions (Dalrymple-Alford et al., 1988). In humans, detailed MRI, PET- scans, CAT scans and other neurological evaluations may provide data concerning transplant growth and viability, but such data do not address the issue of whether the grafts confer long-term benefits to the patient; the only relevant criterion for applica- tion of this procedure.
In this context, not only must the behavioral testing be long- term, it must also be conceived so as to explore multiple aspects of recovery. For example, two studies both examine septohippocampal pathway damage and behavioral effects after cografting (Nilsson et al., 1990; (Cassel et al., 1992c). Nilsson and coworkers found that combined raphe and septal grafts eliminated the spatial memory impairment seen on a water maze task in animals with lesion alone. However, Cassel and coworkers (Cassel et al., 1992c) found that spatial memory deficits in the 8- arm radial arm maze resulting from septohippocampal damage were not alleviated by such cografts. Although we need to be aware that the differing lesion techniques used in these experiments could contribute to differences in resultant behavior, the results above also suggest that behavioral recovery may be seen on one behavioral paradigm, while another closely related one may reveal a continuing deficit.
In a study investigating fetal septal transplants to rats with bilateral fimbria-fornix lesions (Dunnett et al., 1982), behavioral recovery was seen in a rewarded alternation task, but not in spontaneous activity or spontaneous alternation, indicating that observed recovery is task specific. In another study (Dunnett et al., 1985), rats with unilateral ibotenic acid lesion of the nucleus basalis magnocellularis were given embryonic ventral forebrain grafts. A subtle, but important, difference emerged in subsequent behavioral testing. The rats with grafts showed improvement in retention of two tasks (passive avoidance and water maze spatial accuracy), but remained impaired in acquisition of both tasks. Such a difference could indicate problems with anterograde aspects of memory processing. With respect to human testing, we need to be concerned about such task-specific performance. Will uniform recovery be seen on a battery of post- operative tasks which assess motor and cognitive skills? Will some behaviors be improved while others are not, or are actually impaired?
We further need to be concerned about whether the ameliorative effects (when they occur) are long-lasting or only temporary. When synaptic connections with the host brain tissue do occur, are they normal? If so, what are their electrophysiological response properties? What do long-term, evaluative studies suggest as answers to these questions and what relevance do the answers have for clinical models of treatment?
Building on the idea of neurotransmitter replacement or trophic factor release, new techniques, such as genetically engineered cells, are being developed (Horellou et al., 1990). Altered cells, capable of replacement of neurotransmitters, trophic factors, or hormones, could be particularly useful in disease states where specific neural degeneration has led to a critical lack of a specific agent. Although genetically engineered cells avoid some of the problems posed in finding suitable fetal tissue and allow for more specificity of therapeutic agents, there is also the risk of tumorigenicity and the possibility of immune rejection of tissue placed into the compromised brain.
The potential for slow immunological rejection of either tissue grafts or transplanted cells is an important issue. A brief review of this area reveals considerable controversy and doubt. Even with allografts, the concept that the brain is "immunological- ly privileged" is in need of serious revision. It is now thought that, at very best, the brain may be only "relatively privileged". Alternatively, we also need to determine whether the presence of a functionally "ineffective" but growing graft could, at some point in time, lead to detrimental consequences for the patient, such as tumorigenesis or release of toxic byproducts.
If, after all the risks and benefits are assessed, it is still decided to employ neural grafts, the question of what is the most effective tissue to use in grafting procedures must then also be considered. As can be seen from the experiments above, homologous tissue may not necessarily provide optimal results. With respect to fetal tissue, stage of embryonic development also needs to be considered. Another issue that has received scant attention is the effect of gender differences in recovery from brain damage and whether different courses and types of therapy might be needed for males and females. There are virtually no clinical studies specifically addressing possible differences in male and female response to transplant therapy, despite research showing that the hormonal status of females at the time of brain injury can play a role in determining severity of deficit (Attella et al., 1987), sex-related differences occur in response to CNS injury (Roof et al., 1993a; 1993b; Wan-Hua, 1982; Loy & Milner, 1990), and males and females respond differently to treatment (Gould et al., 1990). The development and use of an expanded battery of motor, cognitive and psychological tests, referred to above, would also serve to elucidate possible dichotomies in gender related response to transplant therapy.
Although neural tissue grafting holds out the promise of potential treatment for a variety of CNS disorders, there are many problems that need to be resolved before the treatment can be routinely applied to human patients. It cannot be stressed enough that, with human patients, the medical, ethical and legal risks involved should be carefully assessed before deciding to proceed with transplant surgery. At present, much more pre-clinical research is needed before the rebuilding of neuronal circuits in human patients is an acceptable and meaningful reality.
William Freed (1990), one of the pioneers in successful transplant work, both with adrenal medulla and neural tissue, sums up our views on the whole subject very precisely:
"An unfortunate consequence of excessive publicity about brain tissue transplantation has been the application of tissue transplantation as though it were a therapeutic procedure. In some cases, tissue transplantation has even been used for diseases other than Parkinson's disease, including schizophrenia, Huntington's chorea, and progressive supranuclear palsy. Tissue transplantation remains an experimental technique and should be applied to humans only in the course of carefully planned therapeutic trials (p. 1434 -- italics ours)."
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Review No. 4: 1. re: page 17, Hodges et al experiment. see page 17, several changes in wording and content have been made. However, regarding the reviewers comments that "it was possible to restore working and reference memory deficits," we believe that our interpretation that the grafts "to some extent improved reference memory ..." is acceptable. See Hodges et al. original paper: "Reduction in "reference" memory errors was not as marked. [as working memory]." 2. The long abstract covers the major points of the paper. 3. Finally, in all references to his name in Volume 82 of Progress in Brain Research, Horellou has two Ls.