Transplantation of embryonic mesencephalic and medullary raphe neurons to the neostriatum of rats with unilateral 6-hydroxydopamine lesions

The implantation of dopamine-rich mesencephalic grafts into the 6-hydroxydopamine (6-OHDA)-lesioned neostriatum of rats was accompanied by marked hyperinnervation by serotonin (5-HT) fibers. The purpose of this study was to examine the possibility that the graft-derived 5-HT hyperinnervation is governed by target-related effects present in the host neostriatum and the question of whether grafts rich in 5-HT cells can ameliorate the drug-induced motor asymmetry resulting from unilateral 6-OHDA lesions. Rats were allocated to one of two groups: lesion plus mesencephalic raphe grafts (group R5-HT/L); and lesion plus medullary raphe grafts (group C5-HT/L). A third group, sham-lesion plus mesencephalic raphe grafts (group R5-HT/S) was included. Complete recovery of (+)-amphetamine-induced rotation was observed only in rats which received 5-HT grafts derived from medullary raphe neurons. There was no marked recovery of apomorphine-induced rotation in either of the R5-HT/L and C5-HT/L groups. Immunohistochemistry showed that the R5-HT/L and C5-HT/L groups had 5-HT hyperinnervation in the neostriatum of the lesioned side. There was no target-related effect of the 6-OHDA-lesioned neostriatum specific for the different types of 5-HT tissue. It seems likely that the 5-HT tissue derived from the medullary raphe may contain additional neurotransmitters which contribute to the behavioral recovery.     

Tryptophan hydroxylase inhibition increases preprotachykinin mRNA in developing and adult medullary raphe nuclei.

In order to study the regulation of co-localized monoamine and peptide neurotransmitters in the medullary raphe nuclei (MRN), we determined whether inhibition of serotonin (5-HT) synthesis affected levels of preprotachykinin (PPT; the prohormone precursor of substance P) mRNA in the MRN. Adult rats received p-chlorophenylalanine (pCPA), an irreversible inhibitor of tryptophan hydroxylase (TPH), via Alzet minipumps. TPH activity was inhibited by 70-80% for 3 weeks following pump implantation. During this period Northern mRNA analysis revealed that PPT mRNA levels in the MRN were increased 1.5-2-fold. The pCPA-induced increase was specific for PPT mRNA since no change was detected in mRNA coding for neuron-specific enolase (NSE; a constitutive neuronal protein) or 28 S ribosomal RNA. To determine whether fetal inhibition of 5-HT synthesis affected development of PPT mRNA in the MRN, pregnant rats were administered pCPA via Alzet minipump implanted on embryonic day 8. In pCPA-treated litters TPH activity was decreased by 60-70% from E16 to postnatal day 3 (P3), returning to control levels by P8. Northern mRNA analysis revealed that PPT mRNA levels increased 2.4-fold of control levels at P1. Infusion of pCPA for one week resulted in an earlier increase in PPT mRNA levels, suggesting that birth was not required to elicit the surge in PPT message. These results support the hypothesis that alterations in 5-HT metabolism have regulatory consequences for co-localized substance P formation in the MRN.     

Implantation of genetically modified mesencephalic fetal cells into the rat striatum.

Transplantation of dopamine (DA) cells into the rat model of hemiparkinsonism induced by intranigral 6-hydroxydopamine (6-OHDA) injections has so far focused mainly on DA replacement via a pump-like mechanism. In the present study, we employed a model of hemiparkinsonism that uses an intrastriatal approach to lesioning the nigrostriatal DA pathway to assess the possibility of using cell transplantation to cause regeneration of that system. Toward that end, we transplanted two types of cells on the side of the 6-OHDA-induced lesions: 1) nonmodified fetal mesencephalic cells and 2) fetal mesencephalic cells that have been infected with a retrovirus vector containing a PKC beta 1 cDNA. Both types of cells cause behavioral improvement although the changes were more prominent and occurred earlier in the PKC-modified groups. Tyrosine hydroxylase (TH) immunocytochemistry revealed significantly cell survival in both groups of animals; in situ hybridization studies confirmed the continuous expression of TH mRNA in both groups. Interestingly, long TH-positive axons were observed only in the striata of animals implanted with PKC-modified cells. More importantly, surviving endogenous nigral TH-positive cell bodies were found only on the lesioned side in the latter group. The observations in these animals were associated with significantly smaller decreases in [3H]mazindol-labeled DA uptake sites in both the striata and substantia nigra pars compacta on the side ipsilateral to the 6-OHDA-induced lesions. Furthermore, immunohistochemical studies revealed increased gliosis in the striata of animals grafted with the PKC-modified cells. When taken together, these results indicate that transplantation of normal fetal mesencephalic cells can cause behavioral improvement by providing DA to the host striata whereas PKC-modified cells can, in addition, prevent the progressive degeneration of or cause regeneration of the dying nigrostriatal DA neurons in this model of hemiparkinsonism. These results are discussed in terms of their support for a role for second messenger systems and glial cells, as well as extracellular matrix molecules in the regeneration of the CNS.     

Structural and functional analysis of raphe neurone implants into denervated rat spinal cord

The ability of grafts of embryonic raphe cells to the adult rat spinal cord to reverse the morphological, neurochemical and functional deficits caused by ablation of the serotoninergic afferents has been studied. After grafting, extensive reinnervation was observed by 5HT-immunoreactive fibres, many of which appeared to make contacts with host motoneurones. The neurotransmitter complement was apparently normal. There was also a reinstatement of 5HT levels in the denervated cord after transplantation, amounting to some 40% of normal at the level of the graft. Similarly, Na+-dependent uptake of [3H]-5HT into P3 fractions was over 40% of that recorded in unlesioned animals. Antidromic stimulation of the ventral roots of the spinal cord was used to assess the degree of motoneurone excitability. The field potential in the ventral horn of grafted animals was increased by electrical stimulation of discrete regions along the cord, probably corresponding to the graft loci. It is concluded that serotoninergic neurones transplanted to the denervated spinal cord survive and develop normally, reinnervating the host tissue extensively. Furthermore, the graft/host connections appear to be functionally viable.     

Transplantation of fetal spinal cord tissue into the chronically injured adult rat spinal cord

Transplants of fetal central nervous system (CNS) tissue into the acutely injured rat spinal cord have been demonstrated to differentiate and partially integrate with the adjacent host neuropil. In the present study, we examined the potential for applying a transplantation approach to chronic spinal cord lesions. In particular, we were interested in learning whether host-graft fusion would be adversely affected by an advanced histopathology characterized in part by glial scar formation. Hemisection cavities were prepared at lumbar levels of the adult rat spinal cord 2-7 weeks prior to the transplantation of spinal cord tissue obtained from 14-day rat fetuses. Graft survival, differentiation, and integration with the host spinal cord were subsequently evaluated by light microscopic techniques at post-transplantation intervals of 1-6 months. Immunocytochemistry was also employed to examine the extent of astrocytic scar formation at the host-graft interface and serotoninergic innervation of the grafts. In some other cases, anterograde and retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase was used to determine whether axonal projections were formed between the host spinal cords and grafts. By 2 weeks after injury the initial lesion cavities were surrounded by a continuous astrocytic scar which remained intact for at least 7 weeks after injury in nongrafted control animals. In other animals, transplantation into these advanced lesions resulted in well-differentiated grafts with a 90% long-term survival rate. Although dense gliosis was still present along the lesion surfaces of the recipient spinal cord, foci of confluent host-graft neuropil were observed where interruptions in the scar had occurred. Donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. Thus, some regions of confluent graft and host neuropil could be routinely identified, despite the presence of a dense glial scar along the walls of the chronic lesion site at the time of transplantation. Anterograde and retrograde tract-tracing results suggested that some axonal projections into these grafts had originated from host neurons located immediately adjacent to the donor-recipient interface. In addition, immunocytochemistry revealed some host serotoninergic axons (presumably of supraspinal origin) traversing nongliotic interfaces. The results of this study raise the possibility that grafted fetal CNS tissue has a capacity for stimulating partial regression of an established glial scar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autologous adrenal medullary, fetal mesencephalic, and fetal adrenal brain transplantation in Parkinson's disease: a long-term postoperative follow-up.

We report on the clinical status of 5 patients with Parkinson's disease (PD) 3 years after autologous adrenal medullary (AM)-to-caudate nucleus (CN) implantation, and of 2 PD patients, 2 years after fetal ventral mesencephalon (VM)- and fetal adrenal (A)-to-CN homotransplantation. Current clinical evaluation of 4 of the AM grafted patients revealed sustained bilateral amelioration of their PD signs, most notably of rigidity, postural imbalance and gait disturbances, resulting in a substantial improvement in their quality of life. The disease-related dystonia of one of them disappeared only 2 years after surgery. The levodopa requirements of 2 of these patients and the anticholinergic therapy of another have been reduced. In agreement with the satisfactory clinical evaluation of these 4 patients, their neuropsychological and electrophysiological improvements, initially registered 3 months after surgery, have been maintained for 3 years. After 1 year of significant recovery, the 5th patient of this group has almost returned to her preoperative state. The 2 homotransplanted patients also showed sustained bilateral improvement of their PD signs. Two years after surgery, the most improved signs of the fetal VM case were rigidity, bradykinesia, postural imbalance, gait disturbances and facial expression. The fetal A case has only shown amelioration of rigidity and bradykinesia. Neither of them has shown significant neuropsychological changes. Their current levodopa requirements are less than before surgery. The improvements shown here by PD patients after brain tissue grafts go beyond those obtained using any other therapeutic approach, when levodopa fails. Although more studies and the development of these procedures are obviously required, these initial human trials appear to be resisting the test of time.     

Transplantation of fetal rat cortex into regenerating nerve to the biceps femoris of adult rat

Morphological aspects of the implantation of 11-day-old fetal (E11) rat cortex into regenerating peripheral nerves of adult rats were analyzed during the first 3 weeks after transplantation. On day 11 of gestation, donor fetuses were removed from one uterine horn of pregnant Sprague-Dawley rats and pieces of cortex (1.0 X 0.5 mm) were dissected from them for transplantation. Fetuses and normally delivered pups from the other uterine horn were used as age-matched controls. In 45 adult male Sprague-Dawley rat hosts (200 to 300 g), the epineurium of the nerve to the biceps femoris muscle was crushed and incised, the perineurium minced, and E11 donor cortex was injected into the nerve. At 1 to 7 days postimplantation (DPI), undifferentiated neuroepithelial cells were dispersed or in oval neuroepithelial-like structures throughout the regenerating nerve. At 7 to 14 DPI, neurons and neuroglia were differentiating and interspersed among fascicles of regenerating host axons. At 14 to 21 DPI, aggregates of differentiated neurons surrounded by neuropil containing a rich synaptic bed were interspersed with regenerated host peripheral axons. Fetal cortical implants survived, increased in size, and differentiated throughout the postoperative period, matching their timed controls.     

Morphology and fine structure of the feline neonatal medullary raphe nuclei.

A light and electron microscopic study of the caudal medullary raphe nuclei of neonatal kittens reveals that these nuclei are composed of three size classes of neurons with several possible subclasses. Internuclearly projecting dendritic arborizations in the transverse plane and intranuclear projections in the sagittal plane are common features of large and medium size class neurons of raphe nuclei magnus and obscurus though not for the cells of nucleus raphe pallidus. A positive correlation exists between neuron size and density of axosomatic and axodendritic synapses, which suggests that the large class neurons are the first to receive input in synaptogenesis, which is occurring at this time. A wide variety of synaptic forms and integration is also a characteristic feature within these nuclei, though it is not clear whether this morphological variance represents a phylogenic and/or ontogenic trend or just an expression of the multifunctional nature of this region.     

Serotonin turnover in discrete hypothalamic nuclei and mesencephalic raphe nuclei of young and adult spontaneously hypertensive rats

Serotonin levels and turnover were analyzed in discrete forebrain and mesencephalic nuclei of young (4-week-old) and adult (14-week-old) spontaneously hypertensive rats and age-matched normotensive control Wistar Kyoto rats. Most changes observed were age-dependent, and occurred only in young, early hypertensive rats. Both serotonin levels and the accumulation rate of 5-hydroxy-tryptophan after L-amino acid decarboxylase inhibition were higher in the nuclei periventricularis and paraventricularis of the hypothalamus of young hypertensive rats than in controls. In addition, 4-week-old spontaneously hypertensive rats showed higher 5-hydroxytryptophan accumulation rates in the nuclei supraopticus and dorsomedialis of the hypothalamus than controls. The only difference in serotonin metabolism found in adult hypertensive rats was high serotonin concentration in the median eminence of the hypertensive animals. Our results suggest the presence of anatomically specific, age-dependent alterations in serotonin metabolism, localized to selected hypothalamic nuclei in young hypertensive rats. These data support a role for the hypothalamic serotonin in the development of the spontaneous (genetic) hypertension in the rat.     

Respiratory failure without pulmonary edema following injection of a glutamate agonist into the ventral medullary raphe of the rat.

Injection of ibotenic acid (IA), a glutamate agonist, into the ventral medullary raphe (VMR; especially the nucleus raphe magnus) of the rat produced respiratory failure and death following a predictable course of events. The response to the IA injection was characterized initially by increased respiratory frequency and was followed by pulmonary arterial hypertension, systemic arterial hypoxemia, acidosis, and hypothermia. Within 90 min apnea occurred as a terminal event in all animals. Gravimetric, bronchoalveolar lavage protein, and histological analyses revealed no evidence of pulmonary edema. Intracerebral (VMR) pretreatment with PPP, a sigma receptor agonist, or scopolamine, a muscarinic cholinergic antagonist, prevented pulmonary failure and death even though postmortem histological analysis showed VMR cell loss and gliosis consequent to the cytotoxic IA injection. Based on the results of the study, it is suggested that the VMR has a role in regulation of pulmonary blood flow. Preliminary pharmacological studies suggested that a disruption of glutamatergic and cholinergic mechanisms mediates the lethal pulmonary phenomenon.     

Dendritic cell recruitment following xenografting of pig fetal mesencephalic cells into the rat brain

Following transplantation into the rat brain, porcine neuroblasts differentiate and integrate host tissue, but due to their xenogeneic nature, these cells are generally rejected within several weeks. This rejection is accompanied by infiltration of the graft by macrophages and alphabetaT lymphocytes, but so far nothing is known about the potential role of dendritic cells (DCs) in this process. DCs are professional antigen presenting cells that have the unique ability to prime naive T cells, thereby initiating an antigen-directed immune response. Here, we provide evidence for DC recruitment following the transplantation of pig mesencephalic neural cells into the striatum of LEW.1A rats, as indicated by the high number of OX62+ cells in the rejecting graft and the absence of V65 staining. DCs were found as early as 3 and 8 days postimplantation together with ED1+ and OX42+ cells. This early recruitment, which is probably due to the surgical procedure, might be a critical step in the rejection process, enabling DCs to be loaded with xenoantigens. The number of intracerebral DCs subsequently decreased, being barely detectable in older non-infiltrated xenografts. However, DCs re-appeared as they were observed in grafts infiltrated by macrophages and T cells, a phenomenon that usually precedes graft rejection. Interestingly, we observed a tight correlation between the number of DCs and that of R7.3+ T cells infiltrating the graft. In addition, DCs were often found in close proximity to alphabetaT cells and most expressed MHCII. Taken together, these findings give credence to a role for infiltrating DCs in the mediation of T cell responses to intracerebral xenografting.     

Tissue culture of human fetal cerebellum: a light and electron microscopic study.

Explants of cerebellar hemisphere from a human fetus of 13 weeks of gestation were cultured for up to 80 days in vitro and studied by light and electron microscopy. In silver-stained preparations, small multipolar neurons and ring-shaped endings suggestive of bouton terminaux were demonstrated. Electron microscopic observations demonstrated morphological development of young neurons, synpases, and astrocytes in cultures. An increase in population and structural complexity of synapses in older cultures indicated the synaptic maturation in vitro. The presence of immature synapses in the starting materal (13 weeks of gestation) by electron microscopy suggests that synapse formation in the human cerebellum begins on or just before 13 weeks of gestation. An extensive migration of young neurons into outgrowth zone was also noted by both light and electron microscopy.     

Significant non-serotonergic raphe projection to the visual cortex of the rat. An immunohistochemical study combined with retrograde tracing.

The present study investigated the distribution of serotonergic and non-serotonergic raphe neurons with direct projections to the visual cortex. The study employed the WGA-apoHRP-Au retrograde transport technique combined with 5-HT immunohistochemical staining. Retrogradely labeled cells were observed in the dorsal raphe nucleus, the median raphe nucleus, and in the B9 and B6 cell groups. One notable finding was the great number of retrogradely labeled, non-5-HT immunoreactive cells. The average percentages of such cells in the various raphe regions were as follows: DR: 52% (n = 401); MR: 35% (n = 311); B9: 24% (n = 129); B6: 95% (n = 200). The present study demonstrated the presence of a significant proportion of non-serotonergic raphe region neurons projecting to the primary visual cortex in the rat. It is suggested that these neurons may complement the aminergic neurons as part of the ascending system which controls the functions of the visual cortex.     

The growth of the feline brain from fetal into adult life. II. A morphometric study of subcortical nuclei

As a continuation of the morphometric studies on the preceding paper, here we report on the rate of growth of the caudate nucleus (n.), thalamus, red n., and the substantia (s.) nigra using, with few exceptions, the same cohort of cats. The same previously used brains (n=64 cats) were allocated to the following age groups: fetal (E) 59 days, postnatal (P) days 1, 7, 15, 30, 45, 60, 90, 120, and 180. Sixteen additional cats, interspersed within the groups, were substituted for the red n. and s. nigra studies. There were six subjects per group (except for E59, n=4). Using a projection microscope and cytochrome oxidase-stained coronal sections, a combined (left plus right sides) total of 4693, 3822, 1636, and 1180 sections were drawn for the caudate, thalamus, s. nigra, and red n., respectively. With computer assistance, the drawings were digitized to calculate mean cross-sectional areas and then the mean volume of each structure per group. The growth time tables for the caudate n., thalamus and s. nigra were fairly synchronous. In terms of percentage of the adult volume, for the left side (both sides grew at a similar rate), the three structures grew at a fast pace between E59 and P30. Thus, at E59 their respective percentages relative to adult volume were 23.7, 29.8 and 22.6% and by P30 the percentages were within adult range (85.2, 115.1 and 87.5%, respectively). Starting at P30, for the thalamus and at P45 for the caudate n., there was a consistent tendency to an overgrow which ranged between 4.3 and 30.9% (at P180, P<0.5) for the caudate and between 0.3 and 15.1% for the thalamus. In addition, starting at P30, the right thalamus tended to be consistently larger than the left by a margin ranging between 0.5 and 11.2% (P120, P<0.05). The red n. grew at a different, slower pace. Starting from a fetal volume equivalent to an 18.6% of adult size, its volume was only a 61.0% of the adult value at P30 and came within range of adulthood size only by P60 (81. 3%). Neither the s. nigra nor the red n. showed any consistent tendency to overgrow or to asymmetry. These findings are discussed in the context of the literature. Furthermore, we discuss general conclusions and considerations pertaining to both papers as well as draw comparisons with the maturational time tables of other developmental landmarks in cats. Finally, in a comparison with growth of human brain structures, we point at the limitations and complexities involved in studying human material and, noting interspecies similarities, we propose that the present data from an advanced gyrencephalic mammal may form the bases for a model of structures maturation in humans.     

Transplantation of cultured fetal spinal cord grafts, grown on a histocompatible substrate, into adult spinal cord

Cell suspensions from 14-day gestation rat spinal cord can be successfully cultured on collagen gels containing laminin. After 7 and 14 days in culture the gels were removed from the dish and slices of gel with cultured cells were transplanted into the dorsal column of adult rats. Thirty days later there was no evidence of the gel. However, grafted neurons with well-developed organelles, axons, dendrites, axosomatic and axodendritic synapses were observed in the white matter of the host dorsal columns. Oligodendrocytes and astrocytes were also observed in the graft. This histocompatible substrate for culturing fetal CNS is a mechanism for transplanting cultured CNS cells as grafts of known maturity and synaptic organization into adult host CNS.     

Fine structure of growth cones in medullary raphe nuclei in the postnatal cat.

Morphological aspects of the dynamic processes of growth cone formation and synaptogenesis have been studied in neonatal kitten (2-17 days) medullary raphe nuclei. The formation and elaboration of dendritic growth cones and primary dendritic trunks is actively taking place on the medium size class neurons (stellates) of these nuclei. The dendritic growth cones are morphologically distinctive due to their population of large dense-core vesicles and postsynaptic position. Another growth cone morphology, interpreted as axonal, is also described. This growth cone is typically found in close association or synaptic contact with the dendritic growth cones and contains, in addition to synaptic vesicles, a dense-core vesicle population distinguishable from that of the dendritic growth cone by the presence of a variety of vesicles containing an eccentrically positioned dense particle. No evidence of axo-axonic or dendrodendritic synapses has been found. Synaptogenesis was found to be occurring on somas, dendrites and dendritic growth cones throughout the medullary raphe nuclei, though this phenomenon was more apparent in indistinctly localized subnuclear spaces termed synaptogenic zones. Within these zones large class neurons are found to have greater densities of both axodendritic and axosomatic synapses than medium and small class neurons respectively. Axodendritic synaptic densities on primary and secondary dendrites of large and medium class neurons are greater than their respective axosomatic synapse densities, which may suggest that the latter forms at a later period of development.