[Neuromethods] Neural Transplantation Methods Volume 36 || Neuroprotective Strategies in Neural Grafting

Neuroprotective Strategies in Neural Grafting

Patrik Brundin and Gabriele S. Kaminski Schierle

1. I N T R O D U C T I O N

The survival of neural grafts is dependent on a multitude of both donor- and host-related conditions. The importance of several of these factors, such as anatomical specificity and age of the donor tissue, surgical technique, immunology, presence of growth factors, and so on, are described in other chapters of this book. The significance of obtaining good survival of neural transplants from a limited source of donor tissue is particularly pertinent when transplanting immature dopamine (DA) neurons. Clinical trials with human embryonic nigral grafts in Parkinson's disease have clearly shown that the implants can provide major symptomatic relief in the patients (Kordower et al., 1996, 1998; Lindvall, 1997; Tabbal et al., 1998). The success of this therapy is intimately coupled to a significant restoration of striatal DA levels, as evidenced by positron emission tomography scans, and this can only be achieved when there is good transplant survival. Also, in experimental animals with lesions of the nigrostriatal pathway, a clear correlation between the number of surviving dopaminergic (DA-ergic) neurons in grafts and the degree of behavioral restoration has been found (for review, see

Brundin et al., 1994). Studies in experimental animals indicate that survival rate of transplanted DA neurons is typically approx 5-15 % (Table 1). Neuro- pathological data from two human transplant cases suggest that the survival rate of grafted DA-ergic neurons in Parkinson's disease patients is in the lower range of this interval (Kordower et al., 1996, 1998). With current clinical transplantation protocols, mesencephalic tissue from a relatively large number of human embryos (4-8 donors per side of the brain) seems necessary to attain valuable therapeutic effects (Lindvall, 1997). Because access to human donor tissue from aborted embryos is limited, clinical neural transplantation programs operate under practical constraints. For neural grafting to become a more widely accessible experimental treatment, it is necessary to improve the survival of the transplanted tissue. The relatively poor yield

From: Neuromethods, Vol. 36: Neural Transplantation Methods Edited by: S. B. Dunnett, A. A. Boulton, and G. B. Baker. © Humana Press Inc., Totowa, NJ

411

412 Brundin and Kaminski Schierle

Table 1 Attempts at Enhancing Survival of Grafted Rat DA-ergic Neurons by Different Treatments

Control Treatment (% Survival) (% Survival) Notes

GDNF

Rosenblad et al., 1996 a 9.7 37.4 Sinclair et al., 1996 b 0.7 8.1

Sinclair et al., 1996 a 1.0 13.0 Granholm et al., 1997 2.5-fold

increase Apostol ides et al., 1997 a 2.4 1.6

Apostol ides et al., 1997 a 2.6 3.4 Mehta et al., 1998 ~ 1.6 2.0 Sautter et al., 19989 5.1 12.9 Yurek et al., 1998 b 1.9 5.8

b F G F Mayer et al., 1993 b 0.9 Mayer et al., 1993 b 1.0

Takayama et al., 1995 a 6.3 Takayama et al., 1995 b 1.5 Zeng et al., 1996 a 10.9

GDNF + b F G F + IGF Zawada et al., 1998 8.2 Zawada et al., 1998 4.1

Lazaroids Nakao et al., 1994 b 15.7 Grasbon-Frodl et al., 1996 b 6.5 Bj6rklund et al., 1997 b 0.9

Karlsson et al., 1999 b

Calc ium antagonists

Finger and Dunnett, 1989

6.4

Kaminski Schierle et al., 1999 b 8.2

Caspase inhibitors Schierle et al., 1999 b 10

1.6 2.7

87.5 20.0 25.0

12.7 6.6

41.4 14.0

2.3

10.9

No absolute quantif icat ion

Fresh Hibernated 6 d Hibernated 6 d

3-wk survival 9-wk survival

24-h survival 7-d survival

Hibernated 8 d Solid grafts in

anterior eye chamber

Increased Dopamine neurons graft volume not quantif ied

21.5

36

Figures are either those stated by the respective authors or have been computed based on data presented in the publication based on one of the two following assumptions:

aApproximately 8% of the cells in dissected embryonic rat mesencephalic tissue are DA- ergic. These assumptions imply that there are around 400,000 cells obtained from each dissected ventral mesencephalon.

b There are approx 35,000 DA neurons in one rat embryonic mesencephalon.

Neuroprotective Strategies 413

of grafted DA neurons has stimulated active research into factors that specifically enhance the survival of grafted DA-ergic neurons. Therefore, this review of neuroprotective treatments in neural transplants will focus on the substantia nigra graft paradigm as a model system. Some of the factors that affect graft survival, and are described in this chapter, are probably relevant to other types of neural transplants, while others are clearly specific for implanted DA-ergic neurons.

First, the chapter describes the time-course of cell death in nigral grafts, the localization of dying neurons within the implants, and to what extent in vitro techniques can be used to mimic cell death in grafts. Second, we briefly discuss why it is important to carefully evaluate the absolute survival rate of DA-ergic neurons in control nigral grafts, when assessing the impact of novel neuroprotective treatments. The obvious pitfall is that if the transplants in the control group, for one reason or another, exhibit unusually poor survival, any observed effects of a neuroprotective treatment are less interesting, and the treatment may not be as useful in clinical trials. Third, mention is made of attempts at improving nigral graft survival that do not entail the addition of a neuroprotective agent to the graft tissue. Thus, this section falls slightly outside the main focus of this chapter, and approaches mentioned here are dealt with in more detail in other chapters, e.g., age of donor tissue (see Dunnett and Bj6rklund, this volume), tissue preparation (see Nikkhah et al., Barker et al., this volume) and including growth factors (see Granholm, this volume). A fourth section speculates what the underlying cellular mechanisms that cause death of transplanted DA-ergic neurons may be. A fifth section elaborates on the positive effects of reducing oxidative stress and its consequences in nigral implants, and a sixth describes data indicating that calcium channel blockers can be neuroprotective for transplanted DA neurons. Finally, the chapter discusses recent results demonstrating that antiapoptotic, caspase inhibitors reduce the death of grafted nigral neurons, but the overexpression of the antiapoptotic protein Bcl-2 does not significantly enhance graft survival.

2. WHEN A N D WHERE GRAFTED DA NEURONS DIE

Until recently, little was known about exactly when and where DA-ergic neurons die in intrastriatal nigral grafts. Several studies have now shown that the majority of DA neurons that die in the grafts do so within the first 1-2 wk following surgery (Nikkhah et al., 1994; Duan et al., 1995; Barker et al., 1996). These conclusions are based on counts of surviving tyrosine hydroxylase (TH)-positive neurons in different graft recipients killed at defined time-points after graft surgery. An alternative theory is that there is


continued death of TH-positive neurons in the grafts over weeks, possibly as part of a normal, regulated, and developmentally programmed cell death (Janec and Burke, 1993), but that this cell death might be balanced by matu- ration of grafted DA-ergic neurons that gradually begin to express the immunohistochemical marker. However, the most likely explanation seems that the majority of dying DA-ergic neurons succumb relatively soon after grafting. Support for this view comes from studies employing markers for dying cells, showing that cell death is most common soon after implantation of mesencephalic grafts. Mahalik et al. (1994) stained nigral grafts with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique, which labels cells with fragmented chromatin. They reported the presence of more TUNEL-positive neurons in 10-15- d-old than in 21-28-d-old nigral grafts. In that pioneering study, there was no description of the grafts with specific cell death markers earlier than 10 d after implantation.

In a slightly different transplantation paradigm, using solid strands of embryonic mesencephalon as donor tissue, Zawada et al. (1998) recently focused on the days immediately after intrastriatal grafting, and demonstrated several TUNEL-positive apoptotic neurons in nigral transplants 24 h after surgery. They calculated that 68% of the grafted DA-ergic neurons died within the first 24 h, and by 7 d 84% were dead. However, in these cal- culations, Zawada et al. estimated that, in the starting donor material, one- half mesencephalon contained only about 4500 DA-ergic neurons. This estimate is low (see below, and the legend of Table 1), and, in reality, the survival of DA-ergic neurons may therefore have been even lower. In another recent study (Emgfird et al., 1999), we utilized the fluorescent stain Fluoro-Jade (Fluoro-Jade, Histochem, Jefferson, AR), which stains cell bodies, dendrites, axons, and terminals of degenerating neurons (Schmued et al., 1997), to label cells in nigral implants. Intensely Fluoro-Jade-stained neurons were relatively numerous at 6 and 10 d after graft surgery, were present in low numbers at 14 d, and had disappeared by 42 d. The same study observed a higher density of Fluoro-Jade-positive neurons in the periphery of the transplants, which may suggest that this is the most unfavorable environment for the grafted neurons (Emg~rd et al., 1999). However, despite there being more dying neurons at the periphery of the implants, it has repeatedly been suggested that the highest density of DA-ergic neurons also reside in peripheral regions (Abrous et al., 1988; Mahalik and Clayton, 1991; Nikkhah et al., 1994), which indeed we were able to confirm in the recent quantitative study (Emghrd et al., 1999). The reason for this phenomenon is not clear, but it is possible that neurons migrate away from the center of


the grafts because of lack of trophic support or poor vascularization, but that, once they have reached the peripheral regions, they still are at high risk of dying.

When exactly is the cell death triggered in nigral grafts? Emerging data suggest that there may be at least two distinct phases of cell death in transplants of dissociated nigral tissue. First, a group of neurons die during the tissue preparation and dissociation itself. A second subpopulation seem to die in the immediate phase after intracerebral implantation. These concepts are supported by data from a study on cultured DA-ergic neurons by Fawcett et al. (1995). They compared the number of surviving DA neurons in cultures of dissociated embryonic nigral tissue, directly after preparation, with the number of surviving neurons in nondissociated solid tissue explants derived from mesencephalon of rats with the same donor age. They observed that tissue dissociation by itself caused the death of about 30% of the DA- ergic neurons. This is similar to our recent observations of cell death, irre- spective of transmitter phenotype, in embryonic mesencephalic tissue, using release of lactate dehydrogenase (LDH) into the medium as a viability index (Fig. 1; Kaminski Schierle et al., 1999; and see Section 8.). Fawcett et al. (1995) found that, after being placed in monolayer culture in vitro, approx 60% of the dissociated DA-ergic neurons were dead after the first day and 87% had died by 3 d after seeding. They attempted to mimic a transplant recipient brain more closely, by culturing the dissociated nigral tissue inside tubes containing collagen gel, and then only an additional 7% of the DA-ergic neurons died over 3 d. A possible conclusion from these experiments is that, although only about one-third of embryonic DA neurons die during the dissociation of the embryonic mesencephalon, there is a continued death once the cells are transferred into a novel environment. The second phase of death may already be initiated by the dissociation trauma, and/or may be exacerbated or triggered if the new environment is not favor- able. Thus, it may be relevant to administer neuroprotective treatments both during the tissue preparation phase and to the graft recipient, during the immediate postoperative phase.

As described above, the cell death caused by tissue dissociation is possible to assess, using, e.g., LDH release (total cell population, Kaminski Schierle et al., 1999) or immunostaining for TH (DA-ergic subpopulation), soon after the cells are seeded in cultures (Schierle et al., 1999a). Both these methods may generate valuable information regarding the potency of neuroprotective treatments. Earlier studies have employed vital stains based on the dye exclusion principle, e.g., a mixture of acridine orange and ethidium bromide (Brundin et al., 1985; Sauer and Brundin, 1991; Nakao et al., 1994;


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Fig. 1. Prevention of cell death and apoptosis in ventral mesencephalic cell suspensions by flunarizine. (A) LDH-release was determined in mesencephalic cell suspensions that were either immediately taken for analysis (0 h) or stored for 6 h at room temperature, plus 2 h at 37°C (8 h), and treated during this time with (open bars) or without (filled bars) flunarizine (1 ~14). Data are expressed as percentage of LDH-activity in the cell supernatant, compared to the initial total cellular LDH- activity at time = 0. (* indicates significant treatment effect, P < 0.001, two-factor ANOVA, F (1, 8) = 57.3; n = 3 in each). (B) Cell suspensions were treated as described above for 8 h, before DNA-fragmentation was determined. (* indicates significant treatment effect, P < 0.001, two-factor ANOVA, F (1, 8) = 339.4; n = 3 in each). Data from Kaminski Schierle et al. (1999).

Barker et al., 1995; Nikkhah et al., 1995; Othberg et al., 1997) or trypan blue (Nikkhah et al., 1995), to assess cell death in embryonic neural tissue used for transplantation. Typically, nigral cell suspensions exhibit viabili- ties above 90% for several hours after preparation. In some cases, it has been possible to detect increased cell viability, using these methods, following neuroprotective treatments (Nakao et al., 1994; Othberg et al., 1997). However, the dye exclusion techniques are marred by the fact that they fail to detect cells that have already lysed, and it is likely that the cell viability is often overestimated. For example, the percentage of viable cells can be very high (>80%) in nigral suspensions, even after several days of tissue storage, even when the suspensions yield poorly surviving grafts (Sauer and Brundin,


1991; Nikkhah et al., 1994). Instead, dye exclusion tests appear to have a more important role in the assessment of cell density in suspensions, and as a means to identify cell preparations that, through inadvertent treatment, exhibit such a low viability that they are unsuitable to graft (Brundin et al., 1985).

3. BASAL SURVIVAL RATE OF GRAFTED DA NEURONS

The reported survival rate of transplanted DA neurons varies greatly from one study to another. First of all, the precise transplantation technique has profound influence on survival. For example, factors such as donor age (Bj6rklund et al., 1980; Brundin et al., 1988; Barker et al., 1995), tissue preparation media (Bj6rklund et al., 1997; Watts et al., 1998a), choice of digestive enzymes (Barker et al., 1995), method and degree of tissue dissociation (Barker et al., 1995; Watts et al., 1998b), inclusion of a centrifugation step (Nikkhah et al., 1994), size of the implantation cannula (Brundin et al., 1990; Nikkhah et al., 1994), and anatomical target of graft injection in the host brain (Bj6rklund et al., 1994) are all likely to affect the survival of the grafted neurons. Although the importance of many of these parameters is self-evident, not all of them have been thoroughly studied. Moreover, methodological factors related to the evaluation of transplant survival may also constitute an important source of variation between different studies. Thus, divergence in tissue fixation protocols, brain section thickness, source of antibodies, and type of cell counting method employed (stereological algorithm [West, 1998] versus Abercrombie [1946] corrected data) should not be overlooked. For example, if the basal survival rate of grafted DA neurons is low in the control group of one study, any neuroprotective treatment found to be efficient in that setting must be interpreted with care. As is summarized in the left column of Table 1, the mean basal survival rate of dissociated DA-ergic grafted neurons, prepared according to routine control protocols, varies by more than a factor of 10 between different studies. A poor survival rate of grafted DA-ergic neurons in the control group may be the result of an inappropriate method of morphological evaluation, or it may represent a truly low survival rate. This, of course, does not necessarily imply that the neuroprotective treatment is uninteresting, but it should be questioned whether it will be equally efficient under other methodological conditions, when the basal graft survival is better.

When calculating the survival of grafted DA-ergic neurons, it is necessary to estimate how many DA neurons were present in the donor tissue at the outset. This is a potentially controversial issue, and there are different approaches to determining how many DA-ergic neurons actually were present in the donor tissue. When using the cell suspension method, there are at least two basic methods to calculate how much nigral tissue has


been grafted. The first method requires that one estimates the number of cells implanted by assessing cell density and noting the volume of tissue implanted in each host. This method assumes that the proportion of DA- ergic neurons in the dissected embryonic mesencephalic tissue has been calculated, e.g., by staining smears of nigral cell suspensions with an anti- body against TH (Sauer and Brundin, 1991). Naturally, this proportion is affected by the size of the dissected ventral mesencephalic tissue pieces, with more liberal dissection margins resulting in a lower proportion of TH immunopositive neurons. Our laboratory typically obtains approx 5-11% TH immunopositive neurons from embryonic rat donor tissue (Sauer and Brundin, 1991) and 9-10% in mouse donor tissue (Nakao et al., 1995; Schierle et al., 1999b). However, even though TH expression is extensive in the substantia nigra of 14-d-old rat embryos (Sauer et al., 1992), it can be questioned whether all the neurons/neuroblasts destined to become DA- ergic actually express TH at this stage. Moreover, this method does not take into account whether there is loss of cells during the preparatory stages, e.g., during mechanical dissociation or centrifugation steps, resulting in a net reduction of donor tissue. Finally, the method is dependent on the cell suspension being rather homogeneous. Otherwise, the sample(s) used to assess cell concentration may not be truly representative of the aliquots of cell suspension that are actually transplanted.

The second method used to estimate the number of DA-ergic neurons in the grafted nigral tissue is slightly easier to apply, and may result in even more valuable information regarding a certain procedure's relevance to clinical transplantation trials. The method is based on knowledge of the number of DA-ergic neurons in the whole mesencephalon of one donor. In laboratory rats, it has been estimated that the mesencephalon contains approx 30,000-40,000 DA-ergic neurons, with about half located in the substantia nigra and the remainder in the adjacent ventral tegmental area (Hedreen and Chalmers, 1972; Swanson, 1982; Bj6rklund and Lindvall, 1984; Fawcett et al., 1995). Mice are considered to contain about half as many DA-ergic neurons per mesencephalon (Lieb et al., 1996), but there are large differences in numbers between different strains of mice (Baker et al., 1980). With knowledge of the number of DA-ergic neurons contributed by each donor, the volume of medium used to mechanically dissociate each piece of donor tissue, and the number of microliters injected into each recipient, one can easily estimate the survival rate of the grafted DA-ergic neurons. Typically, tissue equivalent to one-third to one whole mesencephalon is implanted into each host, also when grafting solid pieces. This method of calculating graft yield is clearly clinically relevant, i.e., when there is


interest in exactly how many DA-ergic neurons survive from a defined number of donors. However, a major shortcoming is that the number of DA- ergic neurons present in the mesencephalon of rats is still not definitely determined using modern stereological methods, and may display strain (Baker et al., 1980) and sex (Beyer et al., 1991) differences.

Another issue when estimating graft survival is the variance in graft size between hosts. It is quite common that there is a 10-fold difference between the largest and smallest graft in a group of animals implanted with tissue from the same cell preparation. Some of this variance is probably inherent to the complex biology of the transplantation procedure, in which immuno- logical factors (if donor and host are not truly syngeneic), marked differences in local brain trauma at the implantation site, and variability in the condition of the neurons within one cell suspension, may all play a role. An important part of the variance is probably derived from factors that are possible to change by altering the methodological protocol. For example, there is less variance in graft size between recipients if the donor tissue is dissociated into a single-cell suspension (Nikkhah et al., 1994), but the dis- advantage with this approach is that the average graft size is substantially decreased, if the donor tissue is excessively traumatized by dissociation into a single-cell suspension (Watts et al., 1998b; and compare yields in Sauer and Brundin, 1991, and Nikkhah et al., 1994).

4. WHY DO GRAFTED DA NEURONS DIE?

The reasons why 90% of grafted DA neurons die are not fully understood. Considering the rather harsh treatment that the cells undergo during tissue preparation and implantation, and that they are moved from the developing mesencephalon to an adult striatal environment, two prime sus- pects emerge as likely initiators of death. As mentioned in Section 2., it seems that about one-third of the cells of the embryonic mesencephalon die during the tissue dissociation step (Fawcett et al., 1995; Kaminski Schierle et al., 1999; Fig. 1), and that a larger proportion progress to death, once the tissue has been injected into the host brain. Several mechanisms that may contribute to the death of grafted DA-ergic neurons are presented in Fig. 2. First, hypoxia, hypoglycemia, and mechanical trauma are likely to play a role. Second, deprivation of appropriate growth factor stimulation may also contribute. Little direct data describes the pathways involved in cell killing in nigral grafts, but a great deal of circumstantial evidence can be obtained by examining results of the studies aimed at improving graft survival, which are described in Sections 6-9.


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Drawing on experience from other areas of related neuroscience research, it can be speculated that oxidative stress and a substantial loss of neurotrophic support are likely to be involved. Hypoxia, hypoglycemia, and brain trauma are considered to lead to complex series of intraneuronal events that can result in an excess formation of free radicals, and loss of outer membrane potential, with concomitant influx of calcium ion (Ca 2+) through glutamate receptor- and voltage-gated channels (see also Fig. 2; Chan, 1996; Lynch and Dawson, 1994; McIntosh, 1993). Ca 2+ may activate several Ca 2+- dependent enzymes, such as proteases, lipases, and endonucleases, which can cause injury to the cytoskeleton, cell membranes, and nucleic acids (Fig. 2; Orrenius, 1989; Leist and Nicotera, 1998b). Increased concentrations of cyto- plasmic Ca 2÷ can also stimulate oxygen-free radical production, because of the activation of phospholipase A 2 and nitric oxide synthase (NOS; Fig. 2; Dawson et al., 1992; Coyle and Puttfarcken, 1993; Lafon-Cazal et al., 1993). Furthermore, changes in cytosolic Ca 2+ levels may cause mitochondria to release factors, e.g., cytochrome-c, that trigger an systematic, energy-dependent breakdown of the cell (Fig. 2; Green and Reed, 1998; Susin et al., 1998). Initiation of an apoptotic process involves, under some circumstances, the activation of a series of caspases, which trigger events leading to cell shrink- age, blebbing of the plasma membrane, dismantling of organelles, condensa- tion of nuclear chromatin, and internucleosomal cleavage of DNA (Fig. 2). Concluding the neuronal demise, the cytosolic contents are packaged into small vesicles, so-called apoptotic bodies, which are phagocytosed by adjacent cells without causing any significant inflammatory response in the tissue. Although the distinction between apoptosis and necrosis is not always absolutely clear-cut in each individual cell, and both forms of cell death may be the result of similar triggers, it is useful to consider both passive (murder) necrotic and active (suicide) apoptotic pathways when searching for strategies to reduce cell death in neural grafts (Fig. 2). As mentioned in Section 2., and as will be described in more detail in Section 9., apoptosis commonly occurs in mesencephal ic transplants (Mahalik et al., 1994; Zawada et al., 1998; Schierle et al., 1999a). In addition, some posttrans- plantation neuronal loss is likely to occur by necrosis. In fact, following various neuroprotective treatments that counteract apoptosis, more than 50% the DA-ergic neurons do not survive grafting. Demise of these cells must occur according to apoptotic or necrotic mechanisms that are insensitive to the antiapoptotic treatments employed.

Most understanding about mechanisms likely to be involved in DA-ergic neuron death comes from cell culture studies. In those, it is apparent that compounds reducing oxidative stress (Frodl et al, 1994; Colton et al., 1995),


glutamate receptor antagonists (Schierle and Brundin, 1999), antiapoptotic agents (Schierle et al., 1999a, 1999b), and various growth factors, e.g., brain- derived neurotrophic factor (BDNF; Hyman et al., 1991; Beck et al., 1993), fibroblast growth factor (FGF) (Beck et al., 1993; Mayer et al., 1993b), and glial-cell-line-derived neurotrophic factor (GDNF; Lin et al., 1993) can all increase the survival of embryonic DA neurons subjected to unfavorable culture conditions. However, it is not possible solely to study cultured embryonic mesencephalic tissue, and thereby identify mechanisms that kill grafted DA-ergic neurons. Although there is some correlation between treatments that protect cultured and transplanted DA neurons, there are also several examples of factors that are efficient in nigral cultures, but not in grafts. For example, BDNF (Sauer et al., 1993; Yurek et al., 1996), neurotrophin-3 (Haque et al., 1996), and secretory FGF (Haque et al., 1995) are ineffective in grafts, but can protect cultured DA neurons (Hyman et al., 1991, 1994; Beck et al., 1993). Presumably, in vitro cell culture paradigms can model the initial phase of donor tissue preparation, but do not as effectively mimic events that take place after the tissue has been implanted into the adult host brain. This second phase is complicated, because, secondary to minor trauma caused by the implantation cannula, the host nervous system may release both factors that promote cell survival, e.g., growth factors and antioxidant enzymes (Azbill et al., 1997; Goss et al., 1997), or mediators of cell death, such as glutamate and free radical species (Lynch and Dawson, 1994; Azbill et al., 1997). Thus, teasing out the details of the cellular mechanisms that mediate death in nigral transplants provides us with a difficult research task for the future.

5. BASIC TECHNICAL MODIFICATIONS IN GRAFTING PROCEDURE THAT PROMOTE TRANSPLANT SURVIVAL

Although this chapter is focused on the addition of neuroprotective agents that improve the survival of grafted DA neurons, this section we will, nonetheless, briefly mention basic methodological modifications that affect nigral graft survival.

Only immature DA neurons survive transplantation. There is an optimal donor age, i.e., beyond a certain embryonic stage the survival rate of grafted DA neurons drops dramatically. If the graft tissue is mechanically dissociated into a cell suspension, the survival of DA neurons is very poor if the donor rat embryos are older than embryonic day (E) 15-16 d (Bj6rklund et al., 1980; Brundin et al., 1988), but, grafts of solid tissue seem to survive well also from slightly older donors (Simonds and Freed, 1990). The most widely used technique is the stereotaxic injection of disrupted mesencepha-


lic tissue into the striatal parenchyma (Bj6rklund et al., 1980; Brundin and Strecker, 1991). The degree of tissue disruption has varied from simply cut- ting the mesencephalic tissue into small pieces (StrOmberg et al, 1989; Watts et al., 1998b) to subjecting the tissue to enzymatic digestion prior to dis- sociating it into a single-cell suspension (Nikkhah et al., 1994) or a mixture of single cells and aggregates (Brundin et al., 1985). The survival rate of grafted DA neurons is lower when the tissue is dissociated into single-cell suspensions (Nikkhah et al., 1994) than when it is only partially dissociated into a mixture of small aggregates and single cells (e.g., Nakao et al., 1994). Watts et al. (1998b) recently specifically compared the survival of DA neurons in grafted embryonic mesencephalon after various degrees of tissue dissociation. They observed improved graft survival and larger grafts when the donor tissue was cut into small pieces, instead of dissociated into a single-cell suspension. However, these results should be interpreted with some caution, because the survival rates of the grafted DA-ergic neurons, regardless of tissue preparation method, were relatively low, compared to many other studies (Table 1).

The composition of the medium used when implanting mesencephalic tissue is also important. A direct comparison of solid nigral transplants to the anterior chamber of the eye found that, when tissue pieces were prepared and grafted in culture medium (Dulbecco's modified Eagle's medium [DMEM]), the survival of DA-ergic neurons was doubled, compared to when a simple balanced salt solution (Hanks' balanced salt solution [HBSS]) was used (Bj6rklund et al., 1997). Similar findings were recently reported by Watts et al. (1998a), who grafted dissociated nigral tissue to the striatum, and obtained more surviving DA-ergic neurons in the transplants when using DMEM, than HBSS or glucose-enriched phosphate buffered saline as medium.

A different approach to trying to improve the survival of grafted DA neurons is the addition of embryonic striatal tissue to mesencephalic transplants, in so-called co-grafts. In most studies (Brundin et al., 1986; Yurek et al., 1990; Costantini et al., 1994; Emghrd-Mattson et al., 1997), there has been an increase in axonal outgrowth, but no increase in the number of surviving DA-ergic neurons. In contrast, some experiments (Sortwell et al., 1998) indicate that embryonic striatal cells can actually increase the survival of grafted DA neurons.

6. T R O P H I C FACTORS AND NIGRAL GRAFT SURVIVAL

Because Granholm (this volume) describes the role of trophic factors in neural grafts in detail, some studies of specific relevance to nigral transplants will be only very briefly described here. Intracerebral infusions, or direct


supplementation of the nigral graft tissue with basic fibroblast growth factor (bFGF), increase graft survival (Mayer et al., 1993a; Zeng et al., 1996). Also, a fibroblast cell line genetically engineered to produce bFGF, which was co-grafted with embryonic mesencephalic DA neurons, was found to increase the survival rate of the DA neurons severalfold in the rat striatum (Takayama et al., 1995). Similarly, several recent studies have shown that GDNF markedly improves the survival rate of grafted DA neurons (Rosenblad et al., 1996; Sinclair et al., 1996; Granholm et al., 1997; Sautter et al., 1998; Yurek et al., 1998). A newly discovered novel member of the GDNF family, neurturin, can also increase the survival of grafted DA neurons (Rosenblad et al., 1999).

Recently, Zawada et al. (1998) chose to combine several growth factors, and incubated solid strands of nigral tissue for 2 h prior to intracerebral transplantation with GDNF, insulin-like growth factor-1 and bFGF. They observed an increase in survival of grafted DA-ergic neurons to about 150% of control, at 24 h after implantation. Pretreatment with the growth factor cocktail still had a similar degree of survival-promoting effect after 7 d, but both the growth-factor-treated and control grafts exhibited a further 50% reduction in DA-ergic cell numbers by then. Notably, after 7 d, each control graft contained a mean of only 723 DA-ergic neurons. Originally, each solid strand graft was derived from one-half mesencephalon, and therefore the basal survival rate was only around 4% in this study (compare with grafts of dissociated nigral tissue in Table 1). Generally, treating the donor tissue with a growth factor, only before transplantation surgery, seems to be less effective than when the factor is administered to the host during the crucial first postoperative week (Mayer et al., 1993a). For example, recent studies indicate that there are, at best, minor (Apostolides et al., 1998), and, in some cases, no (Mehta et al., 1998), effects of GDNF on the survival of grafted DA neurons, when the trophic factor is added to the tissue during a pregraft storage period at 4°C.

7. EFFECTS OF R E D U C I N G OXIDATIVE STRESS A N D ITS CONSEQUENCES IN NIGRAL GRAFTS

Free radicals contain an unpaired electron in their outer shell. Oxidative stress is caused by an imbalance between the production and scavenging of free radicals by antioxidant systems. They are highly reactive and readily damage proteins, DNA, and membrane lipids (Fig. 2; for review, see Floyd and Carney, 1992). When lipids are attacked by radical species, a chain reaction of new radical formation is initiated, leading to disruption of the cell membranes, and ultimately cell death (for review, see Halliwell and


Gutteridge, 1984). Radicals have also been proposed to trigger apoptosis under certain conditions (Murphy and Bredesen, 1998). As mentioned earlier, a likely scenario is that hypoxia and ischemia are evoked in the donor tissue during the transplantation procedure, leading to the generation of free radicals. The resulting oxidative stress may possibly be enhanced by local brain trauma induced at the transplantation site (Lynch and Dawson, 1994; Azbill et al., 1997). Furthermore, nigral neurons may be particularly prone to oxidative stress, because DA can auto-oxidize, or enzymatically be converted to products that generate radicals (Olanow, 1992).

Lazaroids are a family of synthetic compounds that have been proposed to have both antioxidant- and membrane-stabilizing properties (Hall, 1997). Members of the lazaroid family have been found to protect mesencephalic neurons in vitro from serum deprivation (Frodl et al., 1994; Othberg et al., 1997), glutathione depletion (Grasbon-Frodl et al., 1996a), 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine lesions (Sanchez-Ramos et al., 1992), and nitric oxide (NO)-induced toxicity (Grasbon-Frodl and Brundin, 1997). In 1994, we described that lazaroids are also effective at protecting transplanted DA-ergic neurons from death (Nakao et al., 1994). Initially, treatment of the pregnant rat and donor tissue with lazaroid was observed to increase the survival of DA-ergic neurons in dissociated nigral implants to 265% of that seen in nonlazaroid-treated tissue (Nakao et al., 1994). More recently, we found that the survival was increased in similar grafts to about 170% of control, when the donor tissue was only treated by lazaroid during the tissue preparation, without injection of the pregnant rat donating embryonic tissue (Karlsson et al., 1999). Lazaroid treatment can also increase the survival of dissociated DA-ergic neurons grafted after an 8-d storage period at 4°C (Grasbon-Frodl et al., 1996b). In that case, addition of the lazaroid, U-83836E, to the storage medium induced a doubling of the survival of DA- ergic neurons, bringing the survival up to the level of freshly grafted tissue.

The effects of the lazaroid, tirilazad mesylate, which has been tested clinically in, e.g., subarachnoid hemorrhage (Haley et al., 1997), has been examined in solid tissue grafts placed in the anterior chamber of the eye (Bj6rklund et al., 1997). Those authors tested several concentrations, and found that at a concentration of 3 gM tirilazad mesylate increased the survival of the mesencephalic transplants by about 100%. In studies of the two experimental lazaroids, U-83836E and U-74389G, a concentration of 3 gM has been considered toxic, and, routinely, a concentration of 0.3 gM has been used in both in vitro (Frodl et al., 1994; Nakao et al., 1994, 1996; Karlsson et al., 1998) and in vivo (Nakao et al., 1994; Karlsson et al., 1999) studies on embryonic DA neurons. Regarding intraparenchymal transplants of dissociated nigral


tissue, no detailed study has yet addressed the issue of optimal concentration of tirilazad mesylate. Although the experimental lazaroids, U-74389G and U-83836E, seem toxic to cultured DA-ergic neurons at a concentration of 3 ~M (Frodl et al., 1994), that is not the case for tirilazad mesylate when it is utilized in cultures (Othberg et al., 1997), or in intraocular transplants (Bj6rklund et al., 1997). Currently, treatment of graft tissue with tirilazad mesylate at a concentration of 3 ~M is being used in clinical trials with nigral grafts in parkinsonian patients in Lund, Sweden.

Even though lipid peroxidation might play a role in the death of both cultured and transplanted DA-ergic neurons, not all compounds with a docu- mented antioxidant capacity can increase their survival. For example, the spin-trap agents, ~-phenyl-N-tert-butyl nitrone and ot-(4-pyridyl-l-oxide)- N-tert-butyl nitrone (Karlsson et al., 1998), have not been found to improve the survival of grafted DA-ergic neurons. Furthermore, inhibition of NOS, which normally generates NO, and which, in turn, can react with superoxide radical to form highly toxic peroxynitrite (Beckman and Koppenol, 1996), also failed to improve the survival of grafted DA-ergic neurons (Van Muiswinkel et al., 1998). There is evidence that superoxide radicals can play an important role as a source of unpaired electrons, and thereby constitute an early stage in the cell damaging processes that are mediated by free radicals in mesencephalic transplants. Thus, DA-ergic neurons from mice overexpressing the antioxidant enzyme superoxide dismutase, display a fourfold increase in survival rate, compared to neurons from nontransgenic littermates (Nakao et al., 1995). However, adenoviral transfer of the superoxide dismutase gene to embryonic nigral cells had no significant effect on the survival of grafted DA-ergic neurons, possibly because of a low gene transfer rate (Barkats et al., 1997).

Oxidative stress seems to play an important role in the death of grafted DA-ergic neurons. Nonetheless, further research is necessary to define which free radical pathways are most important in the death of transplanted DA- ergic neurons, and at what steps the process can be stopped by addition of antioxidant compounds.

8. Ca 2+ C H A N N E L BLOCKERS

Excessive levels of cytosolic Ca 2+ are detrimental to neurons by activat- ing proteases and NOS (promoting the formation of free radicals) and endonucleases that randomly cleave DNA, and by depolarizing mitochondria, which can lead to the release of proapoptotic factors (Fig. 2). Cytosolic Ca 2÷ homeostasis involves an intricate balance between Ca 2÷ influx and extru- sion over the outer plasma membrane and the storage of Ca 2+ ions in organelles,


primarily in the endoplasmic reticulum and mitochondria (Orrenius, 1989; Leist and Nicotera, 1998b). In addition, several Ca2+-binding proteins, e.g., calbindin-28 and calretinin, can buffer intracellular Ca 2÷ (for review, see Heizmann and Hunziker, 1991). Stimulation of N-methyl-D-aspartate (NMDA) receptors by glutamate, in the presence of glycine, leads to the entry of Ca 2÷ through the receptor-gated channel, which in extreme cases results in excitotoxicity (Fig. 2). Energy depletion, which could be envisaged to occur in embryonic donor neurons during graft tissue preparation, may hamper main- tenance of an outer membrane potential. This can lead to an increased influx of Ca 2÷ ions through the NMDA receptor, influx of Ca 2÷ ions through voltage-gated Ca 2÷ channels, and a reduced efflux of Ca 2÷ via energy-dependent pumps (Fig. 2). NMDA receptors are present in the substantia nigra (Yung, 1998), and there is evidence that excitotoxic mechanisms contribute to the death of cultured mesencepahlic neurons subjected to stress by serum withdrawal, because the NMDA receptor blocker MK-801, at a concentration of 1 and 10 ~M, increases the survival of cultivated DA-ergic neurons (Schierle and Brundin, 1999). However, a 10-~tM concentration of MK-801was not found to enhance the survival of grafted DA-ergic neurons (Schierle et al., 1998), indicating that overstimulation of NMDA receptors does not play an important role in the death of grafted DA-ergic neurons.

The idea of blocking voltage-gated Ca 2÷ channels, in conjunction with neural transplantation, was tested in the 1980s by Finger and Dunnett (1989). They chose to administer the Ca 2÷ antagonist, nimodipine, to the transplant recipients, rather than add it to the nigral cell suspension. They observed greater transplant volumes in hosts treated with nimodipine when the donor tissue was slightly older, from El7 and 20, than is deemed optimal for nigral grafts. Because no specific immunohistochemical technique was employed in that study, it is not possible to determine whether the DA-ergic subpopulation also survived better in the nigral implants. However, nimodipine had no effect on implant volume in rats receiving fresh cell suspensions prepared from tissue of an optimal donor age (El4). Since the grafts in nimodipine-treated hosts contained more blood vessels per unit volume, the authors speculated that the enhanced graft volume was related to an improved vascularization. That study is of particular interest, because it uniquely illustrates the principle that systemic (as opposed to intracerebral) administration of a neuroprotective agent to the transplant recipient may affect the survival of neural grafts, and thereby it focuses on the second phase of graft cell death the authors proposed earlier.

More recently, the authors have demonstrated that a concentration of 1 ~tM flunarizine, a drug found to act as a blocker of L-, T-, and N-type Ca 2÷


channels (Akaike et al., 1989; Tytgat et al., 1988), inhibits death of mesencephalic DA-ergic neurons in serum-free cultures (Schierle and Brundin 1999). Therefore, we tested the effects of 1 ~tM flunarizine on intrastriatal grafts. By adding the compound to the transplant cell suspension, nigral grafts were achieved that contained 2.6x the number of DA-ergic neurons found in untreated controls (Kaminski Schierle et al., 1999). Also, the graft volume was increased by about the same factor, indicating that the neuroprotective effect is not specific for the DA-ergic subpopulation of cells. When we tested the effects of adding flunarizine to nigral cell suspensions that were left at 20-37°C for a few hours after tissue dissociation, to mimic the transplantation protocol, signs of neuroprotection were also detected. There was a decrease in the amount of oligonucleosomal DNA fragmentation (indicator of apoptosis, see Section 9.), and reduced leakage of LDH from dead cells (Fig. 1). The LDH release amounted to approx 40% of the total number of cells in the cell suspension, and may have been the result of necrotic cell death or late-stage apoptosis (in which apoptotic bodies lyse in the absence of phagocytosis). Taken together, these data indicate that several mesencephalic cells die already in the cell suspension, and that part of that cell death may be apoptotic.

The HBSS medium contains only a very low concentration of Ca 2+ (20 J.tM). Therefore, it is likely that flunarizine exerts its neuroprotective effects in embryonic neural cell suspensions also through mechanisms other than blockade of voltage-gated Ca 2+ channels. Indeed, earlier reports have suggested that flunarizine may act as an inhibitor of the mitochondrial mega- channel, which participates in the triggering of apoptosis (Takei et al., 1994; Elimadi et al., 1998). Moreover, it has been proposed that flunarizine can inhibit lipid peroxidation and oxidative stress (Takei et al., 1994). Although it is still possible that flunarizine acts, at least partly, by inhibiting voltage- gated Ca 2+ channels, future studies with drugs that have a more selective profile against Ca 2+ channels may help to clarify this issue. Also, it is of interest to what extent flunarizine rescues the same population of DA-ergic neurons as lazaroids. An optimistic attitude might be that the DA-ergic neurons rescued by lazaroids and flunarizine are not identical, and that the two drugs can produce additive effects in nigral grafts.

9. A N T I A P O P T O T I C A P P R O A C H E S

Neuronal apoptosis is an integral process in the normal development of the nervous system (Oppenheim, 1991; Raft et al., 1993). However, when activated by pathologic stimuli, apoptosis may participate in neurodegenerative disease and cause other undesirable neuronal losses (for review,


see Bredesen, 1995; Leist and Nicotera, 1998a; Pettmann and Hendersen, 1998). A family of caspases (Alnemri et al., 1996) was identified as key exe- cution machinery of this process. The crucial role of caspases for programmed cell death was initially genetically defined in the nematode Caenorhabditis elegans. The first mammalian enzyme found to be homolo- gous to the C. elegans caspase, Ced-3, was interleukin converting enzyme (ICE), now termed "caspase 1". At least 13 more mammalian caspases have been identified (Stroh and Schulze-Osthoff, 1998). Several of these iso- enzymes are found constitutively in neurons as zymogens, and may be proteolytically activated by themselves, by other caspases, or by different proteases. In turn, they activate further enzymes by controlled proteolysis, and cleave or inactivate various vital intracellular structures (for reviews, see Cohen, 1997; Nicholson and Thornberry, 1997). Recently, irreversible enzyme inhibitors mimicking specific cleavage sites have been developed. For example, acetyl-tyrosinyl-valyl-alanyl-aspartyl-chloromethylketone (Ac-YVAD-cmk) corresponds to the caspase recognition sequence in interleukin-l[~. These specific caspase-inactivating peptides effectively block apoptosis in several different experimental paradigms (for review, see Villa et al., 1997). For example, regarding the nervous system in vivo, apoptosis caused by axotomy (de Bilbao and Dubois-Dauphin, 1996), ischemia (Loddick et al., 1996; Endres et al., 1998), excitotoxins (Hara et al., 1997), and trauma (Yakolev et al, 1997) can be blocked by caspase inhibitors.

As mentioned in Section 9., the cell death occurring in nigral grafts is at least in part apoptotic (Mahalik et al., 1994; Zawada et al., 1998; Schierle et al., 1999a). Therefore, interfering with mechanisms that activate programmed cell death could be a possible road to improved graft survival. Recently, the authors found that dissociated embryonic mesencephalic donor tissue was protected by the caspase inhibitor Ac-YVAD-cmk at a concentration of 500 ~tM in cultures stressed by withdrawal of serum from the medium (Schierle et al., 1999a). In a second step, there was evidence of apoptosis in embryonic nigral cell suspensions, detected as activation of caspase 3 and the presence of oligonucleosomal DNA fragments, only a few hours after dissociation of the immature mesencephalic tissue. These signs of apoptosis were almost abolished by adding 500 ~M Ac-YVAD-cmk to the cell suspension. These in vitro experiments suggested that Ac-YVAD- cmk could also be efficient at reducing apoptosis in nigral grafts, and improve the survival of transplanted DA-ergic neurons. Indeed, when the authors subsequently observed a marked reduction in the number of TUNEL-positive, apoptotic cells in nigral implants treated with 500 ~tM Ac-YVAD-cmk, compared to untreated, control grafts 4 d after surgery.


Fig. 3. Histological evidence for increased survival of DA-ergic neurons in nigral grafts treated with the caspase inhibitor, Ac-YVAD-cmk. Photomicrographs show TH-immunostaining of representative coronal sections through the striatum of the host brain receiving control (A) or 500 ~tM Ac-YVAD-cmk-treated (B) grafts. Data from Schierle et al. (1999a).

Finally, the authors compared the survival of DA-ergic neurons in nigral grafts implanted into hemiparkinsonian rats, and also evaluated their impact on a motor deficit in the host. Treatment of the donor tissue with 500 ~tM caspase inhibitor resulted in a 3-4-fold increase in the number of surviving DA-ergic neurons in the grafts (Fig. 3), and, consequently, there was a more rapid onset, and greater degree, of behavioral transplant effects in the hemiparkinsonian rat hosts. This series of experiments highlighted that caspase- dependent cell death is frequent in nigral grafts, but it does not provide an answer to why as many as 90% of DA-ergic neurons die. The survival rate of DA-ergic neurons in grafts treated with the caspase inhibitor, Ac-YVAD- cmk, was still only 30-40%, which can be interpreted in at least two ways. First, the pharmacological inhibition of caspases by the utilized concentration of Ac-YVAD-cmk may not be adequate. Second, there may also be caspase-independent death in the grafts, e.g., necrosis or apoptosis insensitive to the effects of the caspase inhibitor. In the future, it should be possible to find out which of these alternatives is most credible. Ongoing studies


are already examining the effects of caspase inhibitors with other substrate specificity profiles, and the effects of combining caspase inhibitors with, e.g., inhibitors of lipid peroxidation. In future experiments, it may also be possible to ensure a continued delivery of caspase inhibitor to the grafts during the first few critical days when cell death is still ongoing.

In a different recent experiment, we examined whether the continuous presence of an antiapoptotic protein in nigral grafts can improve the survival transplanted DA-ergic neurons. The proto-oncogene, bcl-2 (see also Fig. 2), exhibits great homology to the nematode cell death suppressor gene ced-9 (Hengartner and Horvitz, 1994), and has been identified as a negative regulator of apoptosis in many different experimental settings (for reviews, see Kroemer, 1997; Reed, 1997; Sadoul, 1998). Because the mechanisms of action of Bcl-2 are not fully understood, it is not possible to predict which types of neuronal death will be subject to Bcl-2 regulation. Several lessons regarding the role of Bcl-2 have been provided by experiments using transgenic mice overexpressing human Bcl-2. These mice display less apoptosis following axotomy of peripheral or optic nerve (Dubois-Dauphin et al., 1994; Farlie et al., 1995), cerebral ischemia (Martinou et al., 1994), or expo- sure to certain neurotoxins (Often et al., 1998; Yang et al., 1998). Bcl-2 also seems capable of preventing necrotic death of neuronal cells that is a conse- quence of hypoxia (Shimizu et al., 1996) or increased oxidative stress (Kane et al., 1995; Zhong et al., 1993).

To explore the role of Bcl-2-sensitive pathways in the death of grafted DA-ergic neurons, we recently grafted mesencephalic tissue from mice overexpressing human Bcl-2 to immunosuppressed rats (Schierle et al., 1999b). The bcl-2 transgene was expressed, under control of the neuron-specific enolase promoter, in the mesencephalon already on E13 when donor tissue was harvested. The bcl-2 transgene protected cultivated DA-ergic neurons from death caused by removal of serum from the culture medium and stauro- sporine-induced stress. When embryonic mesencephalic cells were stored in suspension for 8 h in the same type of medium used for transplantation, we observed increased DNA fragmentation and LDH release. However, the bcl- 2 transgene had no impact on either DNA fragmentation or LDH release. In contrast, addition of the caspase inhibitor, Ac-YVAD-cmk, reduced both DNA fragmentation and LDH release. In the neural grafting experiment, there was no effect of human Bcl-2 on the survival of TH-immunopositive neurons, although the Bcl-2 protein was strongly expressed in transgenic grafts 5 wk after implantation (Schierle et al., 1999b). Taken together, the results suggest that cell death in neural transplants involves apoptotic mechanisms, but that these are not negatively regulated by Bcl-2.

432 Brund in and Kaminsk i Schierle

10. C O N C L U S I O N

As evidenced by Table 1, neuroprotection in nigral transplantation has developed rapidly over the past 5 years. Several treatments have now been demonstrated to exert neuron survival-promoting effects, and the underlying cell death mechanisms are beginning to emerge (Fig. 2). The immediate future may see major developments on two fronts: First, the combination of two or more neuroprotective factors may lead to a markedly increased survival rate; second, such a treatment should be possible to apply on human neurons, and thereby drastically reduce the demand on human embryonic donor tissue in clinical transplantation trials.

A C K N O W L E D G M E N T S

Research performed by the authors and reviewed in this paper was supported by grants from the Swedish Medical Research Council, the Thorsten and Elsa Segerfalk Foundation, the Swedish Parkinson's Disease Society, and by a Marie Curie Fellowship within the 4th framework program of the European Commission.

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[Neuromethods] Neural Transplantation Methods Volume 36 || Neuroprotective Strategies in Neural Grafting