Dissecting Embryonic Neural Tissues for Transplantation

Stephen B. Dunnett and Anders Bj~irklund

1. I N T R O D U C T I O N : DONOR TISSUES FOR TRANSPLANTATION

A variety of sources of cells and tissues have been successfully trans- planted into the central nervous system (CNS). Thus, in the first decades of this century came the first reports of successful grafting, not only of CNS tissues (Del Conte, 1907; Dunn, 1917; Le Gros Clark, 1940) and peripheral nerves (Tello, 1911), but also of a variety of nonneuronal tissues, such as skin (Glees, 1940), neuroendocrine cells (Flerk6 and Szentfigothai, 1957), or tissue derived from tumors (Greene, 1943). Nevertheless, most of these techniques were relatively unreliable, and set in a context in which the Zeit- geist (influenced by Cajal's classic studies on degeneration and regeneration in the nervous system [Cajal, 1928]) was that regeneration was essentially minimal in the CNS of mature adult mammals.

The turning point of the modern era came with the unequivocal demon- stration by electron microscopy that local regeneration can take place in the CNS in response to axotomy (Raisman, 1969), and this was closely fol- lowed by reports of successful neural transplantation in rats from three sepa- rate groups (Bjtrklund and Stenevi, 1971; Das and Altman, 1971; Olson and Malmfors, 1970). These demonstrations of principle were rapidly fol- lowed by a series of studies establishing the conditions for reliable transplan- tation of CNS or other tissues into the brain (Das, 1974; Lund and Hauschka, 1976; Olson et al., 1983; Stenevi et al., 1976). One of the principal condi- tions for successful neural transplantation was that CNS tissues must be taken from the brains of donors during a limited time window of embryonic or neonatal development. Neuronal tissues harvested outside the critical time window invariably survived transplantation rather poorly.

As illustrated in subsequent chapters of this volume, the past decade has seen rapid advances in the design and development of alternative cell lines and engineered cells for transplantation. Nevertheless, for many experimen- tal (and therapeutic) purposes, the most effective graft survival, integration,

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

3

4 Dunnett and BjOrklund

and functional repair are still achieved by using primary embryonic neurons harvested at a critical embryonic age, when the cell has a maximal capacity for growth and plastic development. The selection, collection, dissection, and handling of primary embryonic neuronal tissues is therefore the topic of this first chapter.

2. CRITICAL EMBRYONIC AGES

2.1. Empirical Principles

In contrast to grafts of peripheral nervous system origin that can survive from postnatal, adult, or even aged donors, grafts of primary CNS neurons only survive well if taken from embryonic (or, in exceptional cases, neo- natal) donors. This principle has been established empirically. In particular, Olson et al. (1983) have used the intraocular transplantation model (see Strrmberg, this volume) to provide the most extensive mapping of the criti- cal period for a wide variety of neuronal populations of experimental interest, based on the empirical criteria of relative survivals of each tissue harvested from donors at different embryonic and neonatal ages (see Table 1). Studies using other transplantation methods essentially yield similar values.

The critical period during embryonic development, when CNS tissue will survive transplantation, is different for each particular population of neu- rons. Thus, good survival is only achieved if the tissue is taken for transplan- tation during a limited time window in early development, which, in rats, may be as tight as 1-2 d of duration; the precise timing of that critical win- dow varies from one population of neurons to another. For example, brain- stem catecholamine neurons, such as dopamine (DA) cells of the substantia nigra, noradrenaline cells of the locus ceruleus, or serotonin cells of the central raphe, survive best when grafted from relatively young donors at approx embryonic d 14 (El4) (Table 1). Septal or striatal cells survive best from embryos a day or more older; cells of the neocortex and hippocampus survive best from embryos later in gestation. Indeed, some cortical and hippocampal neurons can survive even from donors taken in the first days of postnatal life.

2.2. Theoretical Principles

The optimal donor ages have initially been determined empirically, but the critical time window corresponds to the period when the individual popula- tions of cells are born during development. Birthdates can be determined from descriptive studies relating to when a particular phenotype is first expressed (e.g., mapping out the appearance of catecholamine fluorescence or tyrosine hydroxylase [TH] expression in the developing ventral mesencephalon [VM]

Dissecting Embryonic Neural Tissues

Table 1 Optimal Ages and Stages for Transplanting Embryonic Rat Tissues

Gestation CRL Carnegie Expected Region day u (mm) u stage b growth (%)'~

Parietal cortex 17-19 18-24 F 200-500 Entorhinal cortex 15-19 14-25 21-23 200-400 Hippocampus 19-21 26-34 F 200-600 Dentate gyrus 20-22 30-36 F/N 300-600 Cerebellum 14-15 11-13 19-21 400-800 Olfactory bulb 17-19 19-25 F 0-100 Spinal cord 15-17 14-20 21-23 100-400 Caudate nucleus c 15-16 12-16 20-22 200-300 Septum ~' 15-16 12-16 20-22 0-100 Substantia nigra c 14-15 10-14 18-21 100-200 Locus ceruleus" 14-15 10-14 18-21 50-200 Dorsal raphe" 14-15 10-14 18-21 100-200

aBased on Table II in Olson et al. (1983), including all data on expected % growth follow- ing transplantation in oculo. The day following mating is defined as d E0.

b Carnegie stages based on Butler and Juurlink (1987); F, fetal; N, 1-2 d postnatal. " The authors' present experience of monoamine-rich intracerebral grafts indicates that the

optimal age for these tissues is somewhat younger than suggested in the original account of Olson et al. (1983), and the day, CRL, and stage data are corrected accordingly. The compara- tive data on growth remains based on their in oculo experiments.

Reproduced with permission from Dunnett and BjOrklund (1992).

[Olson and Seiger, 1972; Specht et al., 1981]), or by explicit labeling of dividing cells, using injections of a DNA marker such as thymidine or bro- modeoxyuridine. Following this latter strategy, Altman and Bayer have pro- duced, over the past 30 yr, a systematic series of autoradiographic studies based on incorporation of [3H]thymidine into developing neurons through- out embryonic, fetal, and postnatal stages of development. A synthesis of these studies has recently been published as a systematic summary of neu- ronal ontogeny in their Atlas of Prenatal Rat Brain Development Altman and Bayer, 1995). This atlas provides a valuable reference for the ages of neuronal mitosis throughout the neuraxis and across the whole period of brain development in the rat. By taking into account an approx 1.5-d differ- ence in developmental age between rat and mouse during the second half of gestation, these data can be used effectively for determining the critical developmental time windows for neuronal populations for transplantation in mouse also.

There may be several reasons for the correlation between the critical donor age for effective survival of neuronal grafts and the birthdates of those neurons.

6 Dunnett and Bjf~rklund

At this age, the fate of the cells is determined, and they have undergone final differentiation; the cells are small and still rounded, so they will be less susceptible to trauma than a more mature neuron; immature cells are able to sustain more prolonged anoxia than mature cells. At the same time, newly differentiated cells are commencing the period of rapid phenotypic matura- tion, neurite extension, and axon growth to distant targets, under the control of the cells' own ontogenetic programs. Certainly cells taken any later in development survive less well, which may be the result of their greater sus- ceptibility to axotomy and anoxia, and a progressive decline in plasticity. Conversely, if cells are taken any earlier, at the progenitor cell stage, they subsequently show only limited capacity to differentiate into the variety of neuronal phenotypes of experimental interest. This would suggest that the signals that regulate and sequence differentiation in the developing embryo are not fully reproduced in the more mature brain (see Chandran and Svendsen, this volume).

2.3. Practical Issues

When designing a novel transplantation program, the first issue to be encoun- tered is determining when the critical time window occurs for any popula- tion of donor cells to survive. Several alternatives are immediately apparent.

2.3.1. Previous Transplantation Studies

If the cells or tissues of experimental interest have been successfully transplanted in previous studies, the most direct method of determining criti- cal donor age is to review the relevant previous literature (Brundin and Strecker, 1991; Dunnett and Bj6rklund, 1992; Olson et al., 1983; Seiger, 1985). Even if the transplantation protocols or experimental purposes are very different, the systematic empirical studies of a wide variety of tissues in in oculo grafts frequently provides good initial data (Table 1).

2.3.2. Previous Tissue Culture Studies

The viability of CNS neurons in tissue culture is similarly dependent on donor age. The large culture literature can frequently provide clues, not only about critical ages and stages of development for optimal survival, but also about other handling issues, such as the susceptibility of particular popula- tions of cells to different media, growth factors, or neuroprotective agents (Shahar et al., 1989).

2.3.3. Studies of Neuronal Birthdates

In the absence of specific studies of the survival of isolated embryonic tissues, good developmental clues as to optimal donor age can be derived from studies of neuronal birthdates, as determined by thymidine or bro-

Dissecting Embryonic Neural Tissues 7

modeoxyuridine incorporation into dividing cells. The most systematic data available for the rat is in the summary atlas of Altman and Bayer (1995).

2.3.4. Studies of Neuronal Differentiation Direct labeling at the time of cell division is the most reliable index of neu-

ronal birthdates, but more conventional descriptive anatomy and immuno- histochemistry can provide detailed descriptions of the initial appearance and development of critical populations of cells and nuclei in the brain. In particular, what the labeling studies cannot provide is information on the topography of development of particular populations of cells to guide embryonic dissection. Many neurons migrate long distances during devel- opment from their site of genesis (typically in the germinal layers bordering the brain ventricles), so that simply knowing where the target cells finally end up in adulthood is insufficient to guide selective dissection of the rele- vant cells from the embryonic brain. The recent atlas by Foster (1998) pro- vides a detailed longitudinal analysis of the first appearance and subsequent development of eight particular neurochemical phenotypes, based on immu- nohistochemistry with primary antibodies against four catecholamine mark- ers and four neuropeptide transmitters.

2.3.5. Empirical Validation In all but the most robust and well modeled systems, it will always be

experimentally prudent to validate decisions about embryonic age and boun- daries of the dissection in the particular experimental model under develop- ment. Not only may previously published descriptions contain errors, but the basis and accuracy of determining embryonic age can vary (see Staging Embryos, below), guidelines for dissection are essentially descriptive, and hence variable from one experimenter to another, and different transplanta- tion methodologies may impose practical influences that alter the optimal donor age. Thus, for example, graft tissues, prepared as dissociated cell sus- pensions, typically have a narrower critical time window than more robust methods involving whole tissue pieces (see "Basic Transplantation Methods in Rodent Brains" by Dunnett and Bj6rklund, this volume). If the reliability and accuracy of embryonic staging and tissue dissection are to be validated empirically, then specific markers are necessary for the particular target cells, in order to identify their differentiation within transplants, and to quan- tify their rates of survival.

3. STAGING EMBRYOS

Once a critical developmental time window is established for harvesting grafts, the experimenter has the additional practical problem of identifying

8 Dunnett and BjOrklund

Table 2 Estimation of Stage of Pregnancy i n Rats by Palpation Under Ether Anesthesia

Age CRL Carnegie (d) (mm) a stage b Signs at palpation a

4-7 3-5

8-9 6-9

10 10-11 11 12-13 12 8 14-15 13 9 16-17

14 10-11 18-19

15 12-14 20-21 16 15-16 22-23 17 17-19 Fetal

18 21-23 Fetal

19 24-25 Fetal 20 Fetal

22 45 Neonatal

Uterine horns are difficult to find, and have variable thickness.

Uterine horns have small, closely spaced, distinct swellings.

Small, distinct, firm spheres with an increasing diameter,

which approximates the corresponding CRL stage.

Elastic, somewhat ovoid enlargements; width less than CRL.

Fetal structures begin to become palpable; head becomes identifiable; small distinct borders between adjacent fetuses; softer than at d 17.

Fetal indurations appear. Uterine horns are thick, soft, continuous

tubes, if litter is large. Day of birth.

a CRL and palpation signs are based on Table I from Olson et al. (1983) for live embryos in vivo, with the morning following overnight mating defined as E0.

b Carnegie stages at each age are based on Butler and Juurlink (1987). Note that they define the morning of vaginal plug as El, which has been modified to E0 in this table. Also, the CRL measurements given here do not correspond accurately with their report, since the latter were based on fixed tissues.

Reproduced with permission from Dunnett and Bjtrklund (1992).

a suitable protocol for collecting a reliable supply of accurately staged donor

tissue for transplantation, and validating the accuracy of the breeding regimes on offer, whether undertaken in-house or by a commercia l supplier.

The developmental age or stage of a donor embryo can be character ized according to three alternative dimensions (Table 2; Fig. 1).

Dissecting Embryonic Neural Tissues 9

A . Rat B. Mouse

E16

E14

E12 LU

+

E18

• :: ..... : . . . .

8 10 12 14 16 18 20 22

Carneg ie s tage

C • Pig

E32

E30

E28

E26 g m o E24

~ , E22

E E20

E18

E16

E14

E12 , . . . . . . . . , , , , , , , 10 12 14 16 18 20 22

-20

18

-16

2~ 0~

E18 -

E16

g ~ E14

o

f,

El0

-44

=40 E56

-36 E52

E44

-24 ~ ~ E 4 0 - - o

.2° ~ f, ~ E36 -,0g~

- - E3,2 .12 +

-8 + E28

E24

E20

E8

D. Human E60

18

16

9

-12 ~ g

10 ~:~

g 6 g

2

; , i ; , i :, i ~ i i / , i i i o 8 10 12 14 16 18 20 22

Carneg ie s tage

Carneg ie s tage Carneg ie s tage

-32

-28

-24

3 -20 "o

-16 t~

x -12

+

Fig. 1. Relationship between gestational age (filled circles), CRL (open circles), and Carnegie stage of embryos, from four different species: (A) rat, (B) mouse, (C) pig, and (D) human embryos. Based on data provided in Butler and Juurlink (1987) (see also, Table 1).

3.1. Stage

The stage of development is determined by morphological criteria for the development of limbs and organs, and have been described in greatest detail for human embryos, with standard reference to the collections of the Carnegie Institution of Washington, DC (O'Rahilly and Mtiller, 1987). Thus,

10 Dunnett and Bji~rklund

development may be classified into 23 distinct, formal stages in the embry- onic period, during which organs are first formed, followed by the fetal period, when there is further growth and development of those organs. Comparative descriptions are available for other species, including chicks, mice, rats, pigs, monkeys, and humans (Butler and Juurlink, 1987). Nevertheless, accurate staging, based on morphological features of limbs and organs, can be a diffi- cult task, based on subtle judgments, and requiring considerable experience.

3.2. Age

The gestational age of development of an embryo following conception provides the logically simplest measure of embryonic development. How- ever, gestational age alone is not always reliable. First, it can be difficult to determine accurately, unless the time of copulation is observed. Typically, rat matings are determined by overnight pairings and confirmation of copu- lation by the presence of a vaginal plug on the following morning. Neverthe- less, in many species, there can be variability in the time between copulation, fertilization, and implantation, and, in rats, there can be variable rate of devel- opment of embryos with litter size. Note also needs to be taken of variations in practice between different laboratories in designating the day following confirmed overnight mating as either d E0 or d E 1.

3.3. Size

The physical size of an embryo can be determined by the crown-rump length (CRL) (Fig. 2). In practice, this is the simplest measure of develop- ment of the embryo of a particular species. Because of variability in esti- mates of stage and age, CRL should always be given for reference in scientific protocols and experimental reports. Detailed tables for relating these three parameters in the primary laboratory species have been collated by Butler and Juurlink (1987; see Fig. 1).

Pregnant rats for embryo donation can be bred in-house or purchased from approved breeding establishments. There can be considerable variabil- ity in the reliability and accuracy of staged pregnancies from either source. If a breeder does prove reliable and accurate, simply order pregnants of the required age, and monitor accuracy by routinely checking CRL length of the harvested embryos. However, if the breeder is less reliable, the preferred strategy is to order rats several days earlier in pregnancy than required, and then monitor the growth of the embryos in utero on a daily basis, until they reach the required stage of development. This is achieved by estimating the size and morphological features of the embryos by palpation of the mother under a light anesthetic. Although the determination of the size of embryos, based on the cues provided by palpation, is a skill that requires a degree of

Dissecting Embryonic Neural Tissues 11

Fig. 2. Removal of the brain from a 14-mm (El5) rat embryo. 1. Measure CRL, defined as "the greatest length of the embryo as measured in a straight line (i.e., calliper length), without any attempt to straighten the natural curvature of the speci- men" (O'Rahilly and Mtiller, 1987). 2. Kill the embryo by dislocation of the neck (required under UK regulations). 3. Single incision at base of brain, above the eye, using fine pointed scalpel blade (e.g., No. 11). 4. Pry brain open, pinch skin, and peel back. 5. Pry away skin and meninges from over brain snip brain; free at the level of hindbrain.

practice and experience, guidelines indicating the distinctive features that can be detected at each age and stage of rat embryo development have been published (Table 2; Dunnett and Bj6rklund, 1992; Olson et al., 1983).

4. HARVESTING EMBRYOS

Obtaining tissues for transplantation requires harvesting the embryos from staged pregnant rats, removing the brain, and dissection of the relevant tissues. A pregnant rat is taken at the relevant stage of pregnancy, as deter- mined following the procedures of Subheading 3. For most purposes, the pregnant rat is killed by an approved method, then the two uterine horns are removed and placed in a solution of 0.6% glucose-4% saline in a Petri dish. The embryos are then removed, transferred to a second Petri dish, and kept moist with glucose-saline. Alternatively, if the transplantation procedure requires separate embryos at spaced intervals over a period of hours, the dam may be kept alive under deep barbiturate anesthesia, and the embryos removed by cesarean section, one at a time. The dam is then killed at the end of the embryo collection, again by an approved method.

To remove the brains, first kill the embryo by decapitation (as required by UK legislation for embryos beyond halfway through gestation). The brain is

12 Dunnett and Bj6rklund

removed, using a fine scalpel blade and Dumont No. 5 forceps to peel away the overlying skin and cartilage (Fig. 2). The dissection is easier if the dis- secting Petri dish is placed on a bright field light base, so that the embryos are illuminated from below. Then, when visualized in a dissecting micro- scope from above, the tissue is translucent, and the location of the brain is easily seen within the overlying layers of skin and cartilage. The embryonic brains are transferred to a small eyeglass well for dissection, free-floating in glucose-saline.

5. D I S S E C T I N G E M B R Y O N I C G R A F T T I S S U E S

5.1. General Principles

All dissections are undertaken within a standard medium comprising 0.6% glucose-4% sterile saline. The relevant embryonic brain tissues are dissected in the eyeglass wells, with 2-3 mm medium to keep the brain tissues moist. The dissections are carried out using Dumont No. 5 forceps and iridectomy scissors, and sterilized in advance, either by autoclave or by dipping in alcohol and flaming, following standard tissue culture protocols. A dissecting microscope that is provided with both fiberoptic light source and a transmitted light base, to permit illumination from both above and below, can be adjusted to give optimal visualization of the translucent tis- sues within the embryonic brain. When only incident illumination from above is available, the specimen is best viewed against a black background, which helps to bring out details.

Several general principles apply to all dissections (Seiger, 1985):

1. Do not pinch, squeeze, or tear at the target tissue itself. Handle the brain, for example, by lifting, turning, or grasping by the brainstem, rather than by the area to be dissected. Always cut or prise away from the target area, and never directly touch the tissue to be grafted.

2. Keep instruments clean and free from contamination. Maintaining sterility is a worthy goal, but is not an absolute necessity for most purposes, at least in rats.

3. Meninges and adhering mesenchymal tissue will grow vigorously in intracere- bral grafts. It is therefore important to remove these and other nonneural tissues from the neural tissues to be grafted. Separating the meninges from the dissected piece of brain tissue by teasing away is often easier, once the dissection is com- plete, rather than removing all meninges from the surface of the brain (including in all the crevices) prior to dissection. Separation is most easily achieved by grasping a corner of the meningeal sheet with one pair of Dumont forceps and teasing it through the partly open jaws of a second pair of forceps, to trap the neural tissue behind. The separated meninges and graft tissue are easily distin- guished in the microscope: The neural tissue is semiopaque and cream or white

Dissecting Embryonic Neural Tissues 13

in color, depending on the illumination; the meninges comprise a transparent sheet, which is finely vascularized and slightly pink.

4. At the end of the dissection, do not pinch or grasp the dissected piece directly. Rather, lift it between the tips of partly open forceps, or using a loop of bent wire held by surface tension. The tissue is then transferred to a collecting dish (for suspension grafting or further manipulation in vitro), or directly to the host ani- mal (for grafting as solid pieces of tissue).

Embryonic brain tissues are dissected so as to collect the regions of interest for transplantation, drawn from a combination of anatomical knowledge of the relevant neuronal development and empirical criteria drawn from exper- ience and the published reports of what has previously proved most suc- cessful (see Subheading 2, above). Detailed descriptions of dissection of relevant populations of cells from the embryonic brain are available from a variety of sources, including the extensive tissue culture literature (Shahar et al., 1989), and from sources specifically oriented to transplantation (Brundin and Strecker, 1991; Dunnett and Bj6rklund, 1992). The following subhead- ings outline detailed dissections for several of the commonest CNS tissues presently employed in neural transplantation research.

5.2. Ventral Mesencephalon for Dopamine-rich Nigral Grafts The DA-ergic cells of the developing substantia nigra are located in the

VM at the level of the mesencephalic flexure (Fig. 3A-C; Olson and Seiger, 1972; Specht et al., 1981). Most of these neurons are born between E13 and El5 (Lauder and Bloom, 1974), which coincides well with the period when they will survive after transplantation. Very few DA cells survive in suspen- sion grafts from embryos >14 mm, although up to 20 mm is viable (but not optimal) for solid tissue implants (Seiger and Olson, 1977).

The substantia nigra is dissected as follows. The brain is removed and laid on its side (Fig. 3D). The VM is then dissected with the iridectomy scissors, using a lateral approach, as illustrated in Fig. 3D,E. The rostral and caudal coronal cuts can be defined using landmarks on the overlying tectal and thalamic surfaces (Fig. 3D). The lateral cuts are made, first through the uppermost surface of the mesencephalic tube, and then at the lowermost surface (Fig. 3E), to remove a segment of the VM subtending an arc from 4 to 8 o'clock, when viewed in cross section. Peel away the meninges and attached mesenchymal tissue, after moving the butterfly-shaped dissection clear of the rest of the brain.

This dissection should harvest the majority of developing DA cells mak- ing up the substantia nigra and ventral tegmental area in the VM. In the adult rat brain, this area contains approx 50,000 DA neurons. Because the best grafts typically contain 3000-6000 TH-immunoreactive DA neurons,

14 Dunnet t and Bj?Jrklund

B C

Fig. 3. Dissection of substantia nigra from the VM of a 12-mm (El4) rat embryo. Sagittal (A) and coronal (B) sections, and mapping of appearance of TH-immuno- reactive neurons (C), taken from the developmental atlas (Foster, 1998). Schematic drawing of dissection, as viewed from a ventrolateral perspective. (D) Lateral perspective showing the angle of the first two cuts of the dissection, with imaginary lines (arrows) continued to intersect the dorsal surface of the tectum and the thala- mus. (E) Lateral cuts on the upper and lower sections of the mesencephalic tube to cut free the VM tissue. Finally, the meninges need to be prised free from the neural tissue.

one can conclude that only 5-10% of implanted cells actually survive the transplantation process. This figure can be improved 2-3-fold by including anti- oxidants (e.g., lazaroids) or caspase inhibitors in the preparation of the trans- plant tissue (Nakao et al., 1994; Schierle et al., 1999; see Brundin and Schierle, this volume), or by the addition of neurotrophic factors, of which the most potent is glial-cell-line-derived neurotrophic factor (Rosenblad et al., 1996; Sinclair et al., 1996; see Granholm, this volume).

Dissecting Embryonic Neural Tissues 15

The position of the caudal cut will determine how many serotonergic neurons (from the raphe nuclei) will be included in the preparation. Indeed, some serotonergic cells are always present with this type of dissection (Doucet et al., 1989), although they can be subsequently eliminated by incubating the tissue fragments with the toxin 1 ~tM 5,7-dihydroxytryptamine (Doucet et al., 1989).

5.3. Ventral Forebrain for Acetylcholine-rich Septal Grafts Cholinergic cells of the ventral forebrain, containing the developing sep-

tum and diagonal band nuclei, are born predominantly between d El4 and El6 (Semba and Fibiger, 1988), and they can survive transplantation at least up to 17 d of gestation (18 mm). However, there is evidence that functional efficacy is far better when using younger, 14-15 d, embryos (CRL 13-14 mm) (Dunnett et al., 1989).

The standard dissection for ventral forebrain cholinergic neurons con- tains septum and variable proportions of diagonal band (Fig. 4). Place the brain on its dorsal surface, with the ventral surface of the diencephalon upward. Dissect out the septal pieces bilaterally, as illustrated in Fig. 4B,C, using iri- dectomy scissors. Finally, tease off any meninges that remain attached to the septal pieces, especially in the midline.

A few studies have sought to achieve a differential dissection of the devel- oping neurons of the septum and vertical limb of the diagonal band, on the one hand, and of the nucleus basalis and horizontal limb, on the other (Dunnett et al., 1986; Nilsson et al., 1988). This may be achieved by making two coronal cuts to take a coronal slab through the forebrain. The septal pieces can then be dissected from the ventral medial part of the slab and the nucleus basalis magnocellularis (NBM) pieces from the ventral lateral part of the hemisphere, trimming off the overlying striatal eminence. Although the lateral pieces in this dissection clearly contain cholinergic neurons, a reliable differentiation of the cholinergic cells of the NBM from those of the neostriatum, which differentiate in the overlying medial ganglionic emi- nence, has not been established.

5.4. Ganglionic Eminence for Striatal Grafts The striatal primordium develops as two elevations in the rostral floor of the

lateral ventricles, designated the medial and the lateral ganglionic eminences (Fig. 5). Different cell populations are born throughout the second half of the gestational period, but reach a peak at d E14-E15 (Smart and Sturrock, 1979). Different populations of striatal and nonstriatal cells undergo mitosis in the dividing neuroepithelial cell layer bordering the ventricle, and then migrate to settle in, or migrate through, the deeper striatal area. The cells

16 Dunnett and Bj6rklund

®

(9

B ®

, ® ® Fig. 4. Dissection of the septum from a 14-mm (E15) rat embryo. (A) Coronal

sections through the embryonic forebrain, illustrating the level from which the sep- turn will be removed. (B) Ventral view of the forebrain, illustrating the first coronal cut (1) to remove the olfactory bulbs and frontal pole, exposing the frontal tip of the ventricles; lateral cuts (2,3) from the ventral surface of the brain into the brain ventricles, to separate the septum from the ganglionic eminence (see C also), and a fourth cut, coronally, to separate the septum from the bulge of the hypothalamus. A final cut is made in the horizontal plane (C,5) to separate the ventral septal pieces from the overlying cortex. S, septum.

that will develop a DARPP-32-positive medium spiny neuronal phenotype, which comprises the predominant population of striatal output neurons, is believed to differentiate primarily from the lateral ganglionic eminence (LGE); other populations of striatal interneurons originate from the medial ganglionic eminence (MGE) (Olsson et al., 1998).

For dissection, select embryos of CRL 12-16 mm (E15-E16; Table 1). The striatal dissection is illustrated in Fig. 5C-E. Lay the brain on its ven- tral surface, with the dorsal cortex upward; make a longitudinal cut through the medial cortex, and fold aside to expose the striatal primordium in the

Dissecting Embryonic Neural Tissues 17

] seconda ry germinal cell layers

differentiating neurons

Fig. 5. Dissection of striatal eminence from within the lateral ventricular cavity of a 14-mm (El5) embryo. (A,B) The MGE, and LGE, respectively, viewed at two coronal sections, based on drawings in the developmental atlas of Altman and Bayer (1995). (C-E) Successive stages of the striatal dissection to remove the whole gang- lionic eminence, which may subsequently be divided into LGE and MGE pieces (F).

floor of the lateral ventricle; snip off the striatal eminence by a superficial horizontal cut, using the iridectomy scissors. Collect the striatal pieces from both hemispheres.

There are no meninges to be removed in the striatal dissection from the ven- tricular surface. However, the vascular membranes of the choroid plexus will be observed in the ventricles, and should be avoided. The surface of the striatal eminence has a characteristic blotchy appearance that aids identification.

The authors routinely collect the whole ganglionic eminence, comprising both the medial and the lateral elevations of the developing striatal primor- dium, in the same dissections (as illustrated in Fig. 5E). However, there is an ongoing debate about whether a selective LGE dissection will yield a more selective striatal graft (Pakzaban et al., 1993). If the MGE and LGE are to be collected separately, it is easier to divide the whole ganglionic

18 Dunnett and BjOrklund

A. E18 B. E20 C. E22

@

Fig. 6. Dissection of hippocampal anlage from an E20 embryo. (A-C) Develop- ment of the hippocampus at E18, E20, and E22, respectively, based on photomicro- graphs in the developmental atlas of Altman and Bayer (1995). (D-F) Successive stages of the dissection to remove the hippocampus from the inward folded mar- gins of the caudal medial neocortex.

piece after dissection (Watts et al., 1999). Alternatively, if the dissection is to favor just one eminence, then a more restricted dissection is undertaken, leaving the remaining piece attached to the rest of the embryonic brain.

5.5. Hippocampus

The neurons of the hippocampus are born relatively late in embryonic development, with birth of the dentate granule cells extending into the post- natal period (Kromer et al., 1983; Zimmer, 1978).

For grafts that are preferentially rich in pyramidal neurons, select embryos of approx 18-20 d of age (CRL 20-24 mm). The dissection is illustrated in Fig. 6. The hippocampus is located as an inward fold of the caudal medial margins of the neocortex overlying the thalamus. A longitudinal cut is made through the medial neocortex, following the borders of the hippocampus

Dissecting Embryonic Neural Tissues 19

caudally, and the cortex is folded aside (dashed line in Fig. 6D). Make a single cut through the cingulate cortex and underlying fimbria (scissors in Fig. 6E). Prise the hippocampus free of the underlying thalamus, fold backward, and trim free at its temporal pole (Fig. 6F). Collect the hippocampi from both sides, trim free of any excess neocortex, and remove any adhering meninges.

5.6. Neocortex

Like the hippocampus, the neocortex differentiates relatively late in devel- opment, and survives transplantation well, from middle periods of embry- onic development up to several days postnatally. Nevertheless, the extent of growth is greatest from the relatively younger donors (Das et al., 1980). No differences in the phenotypic development of connections have been found among cortical tissues dissected from different areas of the cortical mantle (O'Leary and Stanfield, 1989), although there is some evidence that the topography of the dissection can influence the functional impact of the grafts (Stein et al., 1985).

The most straightforward dissection of cortical tissue involves making a longitudinal cut through medial neocortex, from the frontal to the occipital poles, and folding outward, as in the first stages of the striatal or hippocam- pal dissections (Figs. 5 and 6). The sheet of cortex is then separated from the rest of the brain by a cut along the lateral margin of the striatal eminence (dashed line in Figs. 5 and 6). Because the cortex is generally dissected from relatively older embryos, it is particularly important to remove all meninges.

6. H A N D L I N G GRAFT TISSUES

6.1. Physical Handling of Tissues

Embryonic tissues for grafting are typically fragile and easily damaged. In particular, if the surface of the tissue to be dissected is damaged, not only will this impair the viability of the cells themselves, but it will obliterate the landmarks necessary for accurate dissection. Therefore, a primary concern is to undertake dissections with delicacy, taking care to avoid touching the target tissue itself with the dissection instruments, and using the rest of the brain, meninges, or, indeed, the body of the embryo to keep the tissue anchored.

Cleanliness is necessarily important in order to avoid contamination of the grafts, and using sterile instruments and solutions constitutes best prac- tice. However, in practice, full sterile procedures are not necessary for viable neural transplantation (at least when working with rats), because, as graft hosts, adult rats resist infections well.

20 Dunnett and Bj6rklund

6.2. Media

The authors routinely use sterile glucose-saline for collecting, dissect- ing, and handling graft tissues (Bj6rklund et al., 1983; Dunnett and Bj6rklund, 1992). A number of studies have investigated whether more physiological media would be preferable, but most such studies have concluded that the simple medium is no less effective than any of the more complex alterna- tives and supplements, at least when following standard embryonic dissec- tions and transplantation procedures.

6.3. Storage and Hibernation

For a number of purposes, the ability to store embryonic tissues for trans- plantation at a later stage can add considerable flexibility to surgical pro- tocols. In addition, stored tissues can be treated in vitro with labels, growth factors, or other manipulations, to address a variety of alternative experi- mental or neuroprotection issues. Finally, in a clinical context, storage could allow a battery of safety assessments to be undertaken on the tissues, which could not be achieved within the limited time available, if implanted fresh.

Three basic strategies for tissue storage have been explored: culturing cells for a period in vitro, cryopreservation, and hibernation. Both of the first two alternatives involve a significant loss of tissue viability, and are generally not considered useful for prolonging the availability of primary CNS cells. This does not invalidate the fact that culture procedures can prove useful for expanding populations of dividing cells (e.g., stem cells, immortalized cells and cell lines): These techniques are considered further in Chandran and Svendsen, Mann and Sinden, and Willing and Sanberg, this volume.

The one procedure that has proved most successful for maintaining the viability of primary cells over a period of days is hibernation, in which embry- onic tissue pieces are kept at a low temperature (4-8°C) in a nonphysiologi- cal medium that slows cell metabolism to a low resting level. Tissues can be maintained in the hibernated state for several days before dissociation and implantation with no loss of viability (Gage et al., 1985; Humpel et al., 1994). A detailed hibernation protocol is provided by Saner and Brundin (1991).

7. OTHER SPECIES

The preceding description has focused on the staging, harvesting, and dis- section of developing CNS tissues from embryonic rats. Similar protocols are used for collecting donor tissues from other species; and the authors have established that the present methods work equally well for nigral and striatal grafts from mouse, pig, marmoset, and human donors.

Dissecting Embryonic Neural Tissues 21

All mammalian embryos develop through the same embryonic stages in the same order. The major differences among species occur in the duration of pregnancies and the rates at which embryos develop through the different stages. Essentially, if the critical age and/or CRL is known for optimal via- bility of a particular population of cells in one species (e.g., rat), this can be translated to the critical stage of development according to the Carnegie sequence. This is most easily achieved using the tables in Butler and Juurlink's (1987) Atlas for Staging Mammalian and Chick Embryos (Fig. 1), by turn- ing to the relevant table for the new species (e.g., pig), and looking up the corresponding gestational age and CRL for the same stage of embryonic development. The authors have adopted this approach at the outset of studies of nigral and striatal grafts in pigs and marmosets, based on knowledge of the critical stages in rats, and found the predicted ages, stages, and CRLs fully accurate for selecting critical gestational ages to achieve good graft viabil- ity in the new species.

Secondly, we have to establish the accuracy with which we can stage the pregnancies in the new species, in order to sacrifice the pregnant dam (for nonprimate species) at the appropriate age. The most straightforward proce- dure is timed mating (mice) or accurately timed artificial insemination (pigs), combined with accurate plots of the time course of gestation through the different stages. Marmosets follow a very rigid receptive cycle, so, again, if they become pregnant, the date on which conception probably took place can be accurately predicted (Annett and Ridley, 1992). In practice, and of particular importance for clinical programs of neural transplantation, the most difficult timing of conception and staging of embryonic development occurs with human pregnancies that come to elective termination, and here special procedures are required, to follow ethical as well as practical and biological constraints (Brundin, 1992; Vawter and Gervais, 1998).

Once embryos of other species are obtained at an appropriate stage/age of development, the landmarks for tissue dissection and tissue handling are essentially similar among species. Thus, the authors have found no difficulty in transferring dissection protocols to all other mammalian species, based in each case on familiarity with rats.

Finally, additional safety procedures may be required for handling tis- sues derived from different species, especially if not obtained from recog- nized breeding sources with defined bacterial and virological status. Thus, for example, primate, and, in particular, human, tissues may require handl- ing within category 2 or 3 containment facilities to, protect the experimenter and others from risks of viral infections. In addition, hosts of other species may be more susceptible to infection than are rodents, requiring greater steri-

22 D u n n e t t and BjOrklund

lity in the handling of all tissues, as well as in the conduct of surgical implanta- tion. Finally, al though allograft tissues seldom require immunosuppression, grafting across species barriers adds substantial immunological complica- tions to any transplantation protocol (see Ohmoto and Wood, Watts and Dunnett, and Low et al., this volume).

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

The authors' own studies have been supported by grants from the Medical Research Council and the Wellcome Trust, and the Swedish Medical Research Council.

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