T.J. Collier

FETAL NEURONAL GRAFTS IN MONKEYS GIVEN METHYLPHENYLTETRAHYDROPYRIDINE

Fetal substantia nigra cells of two different gestational ages were successfully transplanted into the brains of three methylphenyltetrahydropyridine-treated monkeys with severe parkinsonian motor and behavioural deficits. Functional improvement continued for 10 weeks after cell grafts into the striata of two monkeys with substantial numbers of tyrosine-hydroxylase-positive fetal neurons at necropsy. Behavioural improvement was correlated with increases in cerebrospinal fluid (CSF) homovanillic acid (HVA) concentrations after the transplants. A control monkey with inappropriately placed transplanted cells of an earlier gestational age remained severely parkinsonian and died during a similar period. CSF HVA fell slightly in this monkey from the low level seen before the transplants. Fetal dopamine neurons of two different gestational ages appear to survive transplantation in primates and have biochemical and functional effects.

Grafting of fetal substantia nigra to striatum reverses behavioral deficits induced by MPTP in primates : a comparison with other types of grafts as controls

SummaryFetal substantia nigra (SN) cells were transplanted into the caudate nucleus (CN) of four vervet monkeys (Cercopithecus aethiops sabaeus) that had been treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP treatment appears to produce a syndrome similar to that observed in patients with idiopathic Parkinsons disease. Normal and parkinsonian behaviors were quantitated by trained observers 5 days/week. Twenty-eight behaviors based on previous factor analyses were individually scored and rated. Parkinsonian signs included freezing, head and limb tremor, difficulty in eating, delayed initiation of movement, poverty of movement, tremor that stopped with intention, decreased response to threats, and lying immobile in the cage. These signs were combined to give an overall rating of parkinsonism. A summary measure of ‘normal’ healthy behavior was also examined, including such behaviors as yawning, scratching, self-grooming, shifting, and eating. Overall ratings of parkinsonism increased and those of healthy behavior decreased after MPTP. In the 4 monkeys grafted with fetal SN cells into the CN, behavior returned to pre-treatment levels by the time of sacrifice (2, 5, or 7.5 months after grafting). Three control subjects were transplanted with either SN cells into an inappropriate brain site (cortex) or inappropriate, non-dopaminergic, cells (cerebellar) into the CN. Subjects were also compared with three control animals that did not receive MPTP but received cryopreserved or fresh SN and other cells into the CN. Only MPTP-treated subjects that received SN cells into the CN showed evidence of a reversal of the MPTP syndrome after transplantation. In addition, grafting in animals that were not MPTP-treated did not appear to affect behavior. This paper reports the specific behavioral effects of severe MPTP toxicity that were or were not reversed after transplantation and suggests that only fetal SN cells grafted into the CN may be able to reverse behavioral deficits in MPTP-treated monkeys.

Transplantation of fetal dopamine neurons in primate brain reverses MPTP induced parkinsonism.

Publisher Summary Parkinsonian signs were produced in the African Green monkeys by the administration of the toxin, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). The transplantation of fetal neurons from the substantia nigra into two monkeys with severe symptoms of hypokinesia, postural and resting tremor, the episodes of movement freezing, and difficulty in initiating movements resulted in a reversal of these symptoms. The functional degree of recovery corresponded with the immunohistochemical observation of tyrosine hydroxylase (TH) positive neurons in the host caudate nucleus and putamen in these two animals. Dopamine metabolite levels in cerebrospinal fluid (CSF) were also elevated in these monkeys. A third monkey with parkinsonian signs received nigral tissue into a control site, the cingulate cortex. This animal failed to show recovery from the toxin-induced parkinsonism. Both early and late gestational tissue survived and showed growth of neurites, which suggested a wide developmental window in the primate brain. The combination of morphological evidence of survival and growth, the elevated neurochemical indices, and the reversal of behavioral motor impairment following the transplantation of fetal neurons into monkeys with parkinsonian signs may provide therapeutic insights into the treatment of Parkinsons disease.

Cryopreservation of fetal rat and non-human primate mesencephalic neurons: viability in culture and neural transplantation.

Publisher Summary The availability, storage, testing, and transportation of donor tissue are significant practical problems associated with the potential use of grafted neural cell replacements in human neurodegenerative diseases. This chapter describes the applicability of cryopreservation on fetal rat and non-human primate mesencephalic neurons that is initially tested on rat ventral midbrain tissue, and subsequently applied to non-human primate midbrain tissue. Direct comparison of fresh to frozen-stored rat tissue in culture and intrastriatal grafts indicates that cryopreservation reduces the number of cells available for use, but those neurons, which tolerate the procedure appear typical in morphology. However, cryopreservation of fetal monkey dopamine neurons yields viable cells in both culture and intrastriatal grafts. While cryopreserved brain tissue yields fewer cells, dopamine neurons that tolerate freeze-storage survive and grow in culture and neural grafts. Thus, freeze-storage of developing brain tissue may provide a means to establish cell banks allowing adequate storage, tissue typing, and transportation of tissue for use in neural grafting.

Peripheral nerve-dopamine neuron co-grafts in mptp-treated monkeys: Augmentation of tyrosine hydroxylase-positive fiber staining and dopamine content in host systems

Previous studies of rats in our laboratory indicate that a molecule or molecules released by Schwann cells exert survival and growth-promoting effects on mesencephalic dopamine neurons. In the present study, we have begun to investigate the potential for Schwann cell augmentation of host dopamine fiber systems and embryonic dopamine neuron grafts in non-human primates. Ten adult male St Kitts African Green monkeys treated with the dopaminergic neurotoxin 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine one year previously, but behaviorally asymptomatic, served as hosts for implant studies. A segment of young adult monkey saphenous nerve was collected to serve as an implanted tissue source of Schwann cell-derived growth factors. Nerve was enclosed in a hollow semi-permeable polymer fiber for implantation into the lateral ventricle, with embryonic ventral mesencephalic tissue co-grafts containing developing dopamine neurons aimed at nearby locations in the caudate nucleus. Control implants consisted of an empty polymer fiber co-grafted with embryonic ventral mesencephalon. Our morphological observations indicate that while no clear augmentation of the morphology of grafted dopamine neurons attributable to co-grafted nerve was observed, this lack of influence may be related to the spatial separation of the co-grafted tissues. In contrast, some monkeys with nerve segments in the lateral ventricle exhibited increased tyrosine hydroxylase-positive fiber staining in the immediately adjacent lateral septal area and the ventricular wall of the caudate nucleus. This enhancement was not associated with empty polymer implants. Levels of dopamine and its metabolite homovanillic acid derived from tissue punches in the caudate nucleus and septal area support the view that monkeys exhibiting morphological enhancement of host dopamine systems also show biochemical increases in dopamine levels and changes in the direction of normalization of the homovanillic acid/dopamine ratio. Biochemical values from a single septal area tissue punch in one animal were an exception to this rule. This study suggests that while the utility of peripheral nerve as a source of dopamine graft augmentation in non-human primates remains to be demonstrated, grafted nerve has a stimulatory effect on host brain dopamine systems in adult, dopamine-depleted monkeys, and that this morphological effect can be dissociated from previously hypothesized injury-induced regeneration.

Matrigel enhances survival and integration of grafted dopamine neurons into the striatum.

Publisher Summary Matrigel is a biopolymer of natural constituents containing large proteins, which have been shown to promote neurite outgrowth in culture. This chapter aims to enhance the survival and promote outgrowth and integration of fetal dopamine neurons transplanted into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rodents immediately after cavitation. The chapter also describes the effects of Matrigel on transplantation of fetal substantia nigra into the striatum. Innervation of blood vessels into the transplanted tissue appeared to be enhanced by the Matrigel. The effects of Matrigel may be because of a number of factors, such as Matrigel contains a large percentage of laminin and type 4 collagens, which are important structural requirements for attachment of cells and neurite outgrowth; or Matrigel may be promoting the survival and outgrowth of the fetal neurons by stimulating growth factors from the endogeneous striatal tissue. The positive effects of Matrigel may be related to promoting ingrowth of blood vessels and thereby increase the circulation in the transplanted region. However, the fact that substance P-positive innervation of the transplanted tissue took the form of woolly fibers, strongly suggests that these fibers are making the appropriate synaptic contacts.

Reversal of Experimental Parkinsonism in African Green Monkeys Following Fetal Dopamine Neuron Transplantation

The motor abnormalities and progressive debilitation associated with Parkinson’s disease has been linked to depletion of the neurotransmitter dopamine in the nigrostriatal system of the brain (12). Supplementation of brain dopamine by administration of the precursor substance L-dopa significantly improves the motor symptoms of the disease, and has been the preferred treatment for the past 20 years (16). However, chronic L-dopa administration often yields negative side-effects (3), and the benefit of the drug diminishes as the disease progresses (7,24).

Biochemical Changes Induced by Embryonic Substantia Nigra Transplanted to the Striatum of Parkinsonian Monkeys

Parkinsonian behavioral deficits are improved following transplantation of fetal dopamine (DA) neurons to the striatum of MPTP-treated monkeys, although the mechanism of the recovery has not been resolved. While there is good evidence for the survival of grafted tyrosine hydroxylase (TH)-positive cells following their placement in caudate nucleus, it is not known whether they provide sufficient DA to the caudate nucleus to account for the behavioral effects of transplantation. Adult male vervet monkeys, which had exhibited stable parkinsonism for one year after systemic MPTP HC1 administration (2 mg/kg i.m. over 5 days) were the hosts. Three of these animals received solid allografts of vervet embryonic substantia nigra stereotactically implanted bilaterally at several sites in the caudate nucleus. One monkey was grafted with the mesencephalon from a 44 day old embryo (age post-conception, assessed by ultrasound measurements). The mesencephalon from a 49 day old embryo was divided between two other monkeys. Gestation is 165 days in this species. Following sacrifice three months later, the brain of each monkey was perfused with chilled saline, removed and tissue punched bilaterally from chilled coronal sections. Tissue punches were frozen in liquid nitrogen until assayed for concentrations of DA, homovanillic acid (HVA), determined by HPLC and GC-MS respectively. The remaining sections of brain were post-fixed and stained for the presence of TH. DA concentration in central and medial substantia nigra of the host monkeys was depleted more than 90% compared to untreated controls (n= 10-13). This magnitude of depletion in the substantia nigra was at least as great as that found in the substantia nigra of severely parkinsonian MPTP-treated monkeys that were not transplanted (n=ll). Thus, the severity of the MPTP lesion in the host monkeys was confirmed. Large numbers of well-defined THpositive fibers were observed in the caudate nucleus of the grafted animals. DA concentrations were raised by up to 50% of untreated control levels in tissue sampled near to the grafts compared to that distant from the grafts (Fig. 1A). At sites proximal to grafts HVA/DA ratios were normalized (Fig. 1B). At positions distal to the grafts, DA levels and HVA/DA ratios were not different from those measured in MPTP-treated monkeys without grafts/1/. DA levels near to the grafts were well within the range observed in asymptomatic MPTPtreated vervet monkeys (greater than 5% of control). The normalized HVA/DA ratios may reflect synthesis and release of DA by transplanted cells in the absence of external host to graft neuronal connections, or may indicate autoregulation of metabolic activity in the grafted cells. These data are consistent with the hypothesis that transplanted embryonic mesencephalic tissue can provide sufficient DA for restoration of the biochemical balance of the dopaminedepleted basal ganglia in primates.

Catecholamine Fibers Form Synaptic Contacts with Hypothalamic Neurons Transplanted Adjacent to the Medial Forebrain Bundle in Rats

The establishment of connectivity between a host brain and a neural graft may be critical to the integration and ultimately the function of the transplanted tissue. It has been well established that neural grafts both receive afferent input from and project efferent fibers to the host.’-’ Few s t u d i e ~ , ~ . ~ however, have examined the issue of connectivity at the ultrastructural level to determine whether transplanted neurons, in fact, receive a relevant synaptic input from host fibers. We have addressed this problem using ultrastructural immunocytochemistry for tyrosine hydroxylase ( T H ) applied to fetal rat hypothalamic grafts that were transplanted adjacent to the medial forebrain bundle (MFB) of adult hosts. The grafted tissue, which contained the supraoptic and paraventricular nuclei as well as other portions of the medial basal hypothalamus, is normally richly innervated by noradrenergic fibers from the MFB. Hypothalamic tissue was dissected from fetal Long-Evans rats (day 17 gestation), and implanted stereotaxically into the brains of young adult hosts according to the previously described protocol used in our laboratory? Following 6-, 12-, and 20-week survival periods, the host animals were anesthetized with sodium phenobarbitol and perfused with 5% acrolein, 4% paraformaldehyde. Sample vibratome sections (50 p m thick) were stained for Nissl substance to locate the grafted tissue. Remaining sections were then processed for preembedded immunocytochemistry and electron microscopy. In addition to those sections stained for TH, some sections were stained for vasopressin-associated neurophysin (NPII) to assess the viability of transplanted vasopressin neurons. Consonant with previous reports,’ most of the grafts appeared healthy and increasingly well integrated with the host brain the longer the survival time. Transplants exhibited striking growth in the host environment, and, within 20 weeks, were several times the size of the original implants. At the light level, NPII-positive neurons and fibers were observed in the grafts as were TH-positive fibers. A dense plexus of TH fibers resembling that found in the supraoptic or paraventricular nuclei was observed in a circumscribed region of one 20-week graft (FIG. 1A). Cell bodies staining for TH, however, were rarely found in the transplants. Ultrastructurally, immunochem-