Noradrenergic Components of Locomotor Recovery Induced by Intraspinal Grafting of the Embryonic Brainstem in Adult Paraplegic Rats.

Intraspinal grafting of serotonergic (5-HT) neurons was shown to restore plantar stepping in paraplegic rats. Here we asked whether neurons of other phenotypes contribute to the recovery. The experiments were performed on adult rats after spinal cord total transection. Grafts were injected into the sub-lesional spinal cord. Two months later, locomotor performance was tested with electromyographic recordings from hindlimb muscles. The role of noradrenergic (NA) innervation was investigated during locomotor performance of spinal grafted and non-grafted rats using intraperitoneal application of α2 adrenergic receptor agonist (clonidine) or antagonist (yohimbine). Morphological analysis of the host spinal cords demonstrated the presence of tyrosine hydroxylase positive (NA) neurons in addition to 5-HT neurons. 5-HT fibers innervated caudal spinal cord areas in the dorsal and ventral horns, central canal, and intermediolateral zone, while the NA fiber distribution was limited to the central canal and intermediolateral zone. 5-HT and NA neurons were surrounded by each other's axons. Locomotor abilities of the spinal grafted rats, but not in control spinal rats, were facilitated by yohimbine and suppressed by clonidine. Thus, noradrenergic innervation, in addition to 5-HT innervation, plays a potent role in hindlimb movement enhanced by intraspinal grafting of brainstem embryonic tissue in paraplegic rats.

The use of animal models to study cell transplantation in neuropathic hearing loss.

Auditory neuropathy (AN) is a form of sensorineural deafness specifically affecting the conduction of the nerve impulse from the cochlear hair cells to the auditory centres of the brain. As such, the condition is a potential clinical target for 'cell replacement therapy', in which a functioning auditory nerve is regenerated by transplanting an appropriated neural progenitor. In this review, we survey the current literature and examine possible experimental models for this condition, with particular reference to their compatibility as suitable hosts for transplantation. The use of exogenous neurotoxic agents such as ouabain or ß-bungarotoxin is discussed, as are ageing and noise-induced synaptopathy models. Lesioning of the nerve by mechanical damage during surgery and the neuropathy resulting from infectious diseases may be very relevant clinically, and we discuss whether there are good models for these situations. We also address genetic models for AN, examining whether the phenotypes truly model the clinical situation in their human counterpart syndromes - we use the example of the hyperbilirubinaemic Gunn rat as a particular instance in this regard.

Surgical techniques influence local environment of injured spinal cord and cause various grafted cell survival and integration.

BACKGROUND: Cellular transplantation to repair a complete spinal cord injury (SCI) is tremendously challenging due to the adverse local milieu for graft survival and growth. Results from cell transplantation studies yield great variability, which may possibly be due to the surgical techniques employed to induce an SCI. In order to delineate the influence of surgery on such inconsistency, we compared lesion morphology and graft survival as well as integration from different lesion methodologies of SCI. NEW METHOD: Surgical techniques, including a traditional approach cut+microaspiration, and two new approaches, cut alone as well as crush, were employed to produce a complete SCI, respectively. Approximately half of the rats in each group received injury only, whereas the other half received grafts of fetal brainstem cells into the lesion gap. RESULTS: Eight weeks after injury with or without graft, histological analysis showed that the cut+microaspiration surgery resulted in larger lesion cavities and severe fibrotic scars surrounding the cavity, and cellular transplants rarely formed a tissue bridge to penetrate the barrier. In contrast, the majority of cases treated with cut alone or crush exhibited smaller cavities and less scarring; the grafts expanded and blended extensively with the host tissue, which often built continuous tissue bridging the rostral and caudal cords. COMPARISON WITH EXISTING METHODS: Scarring and cavitation were significantly reduced when microaspiration was avoided in SCI surgery, facilitating graft/host tissue fusion for signal transmission. CONCLUSION: The result suggests that microaspiration frequently causes severe scars and cavities, thus impeding graft survival and integration.

Grafting of fetal brainstem 5-HT neurons into the sublesional spinal cord of paraplegic rats restores coordinated hindlimb locomotion.

In rodent models of spinal cord injury, there is increasing evidence that activation of the locomotor central pattern generator (CPG) below the site of injury with 5-hydroxytryptamine (5-HT) agonists improves locomotor recovery and restores coordination. A promising means of replacing 5-HT control of locomotion is to graft brainstem 5-HT neurons into the spinal cord below the level of the spinal cord injury. However, it is not known whether this approach improves limb coordination because recovery of coordinated stepping has not been documented in detail in previous studies employing this transplantation strategy. Here, adult rats with complete spinal cord transections at the T9/10 level were grafted with E14 fetal neurons from the medulla at the T10/11 vertebra level one month after injury. The B1, B2 and B3 fetal anlagen of brainstem 5-HT neurons, a grouping that included the presumed precursors of recently described 5-HT locomotor command neurons, were used in these grafts. EMG and video recordings of treadmill locomotion evoked by tail stimulation showed full recovery of inter- and intralimb coordination in the grafted rats. We showed, using systemically applied antagonists, that 5-HT2 and 5-HT7 receptors mediate the improved locomotion after grafting, but through actions on different populations of spinal locomotor neurons. Specifically, 5-HT2 receptors control CPG activation as well as motoneuron output, while 5-HT7 receptors contribute primarily to activity of the locomotor CPG. These results are consistent with the roles for these receptors during locomotion in intact rodents and in rodent brainstem-spinal cord in vitro preparations.

Brain stem cells adopt a pituitary fate after implantation into the adult rodent pituitary gland.

Fetal brain stem cells (RSCs) have been induced to express pituitary phenotypes in vitro in co-cultures with GH(3) cells and by exposure to GH(3)-conditioned media. In the current studies, we graft RSCs into the pituitary glands of adult rat to investigate whether grafted RSCs can be induced by the native gland to acquire pituitary properties. Grafted cells survive for 4 weeks and express Pit-1, GH, FSH, LH, ACTH, TSH and to a lesser extent PRL indicating that inductive influences are operative in vivo as well. This demonstrates that pluripotential cells can be induced to acquire properties of tissues different from their organ of origin likely through the action of cell-cell contact and local tissue factors.

Regeneration and recovery of the hearing function of the central auditory pathway by transplants of embryonic brain tissue in adult rats.

The present study is the first report of successful regeneration and recovery of hearing function of the central auditory pathway after transection in the adult rat. The ventral cochlear tract in the brain stem to pons was transected on one side in adult rats. Tissue from embryos (E14 to E16) was used to cover the lesion site. In 30% of the rats examined, the axons regrew beyond the transected site and regenerated into the denervated side and terminated at the normal targets. The hearing function of rats was elucidated by recording the auditory brain stem response (ABR). Rats with successful regeneration showed nearly normal ABR. In rats receiving simple transection without covering embryonic tissue, there was no regeneration and hearing function did not recover. Thus, the present findings contradict the widely held view that the adult mammalian central auditory system cannot be restored following damage.

Patterning signals acting in the spinal cord override the organizing activity of the isthmus.

The regionalization of the neural tube along the anteroposterior axis is established through the action of patterning signals from the endomesoderm including the organizer. These signals set up a pre-pattern which is subsequently refined through local patterning events. The midbrain-hindbrain junction, or isthmus, is endowed with such an organizing activity. It is able to induce graded expression of the Engrailed protein in the adjacent mesencephalon and rhombencephalon, and subsequently elicits the development of tectal and cerebellar structures. Ectopically grafted isthmus was also shown to induce Engrailed expression in diencephalon and otic and pre-otic rhombencephalon. Fgf8 is a signalling protein which is produced by the isthmus and which is able to mimic most isthmic properties. We show here that the isthmus, when transposed to the level of either rhombomere 8 or the spinal cord, loses its ability to induce Engrailed and cerebellar development in adjacent tissues. This is accompanied by the down-regulation of fgf8 expression in the grafted isthmus and by the up-regulation of a marker of the recipient site, Hoxb-4. Moreover, these changes in gene activity in the transplant are followed by a transformation of the fate of the grafted cells which adjust to their novel environment. These results show that the fate of the isthmus is not determined at 10-somite stage and that the molecular loop of isthmic maintenance can be disrupted by exogenous signals.

Regeneration of the central auditory pathway in adult rats.

Regeneration of the auditory pathway in the central nervous system, which has not been reported previously, was investigated in adult rats. The ventral cochlear tract (VCT) in the medulla oblongata of adult rats was completely transected unilaterally by a ventral approach through an opening in the basioccipital bone. After postoperative intervals of 1 day to 3 months, the cochlear tract was examined by an anterograde tracing method using wheat germ aggulutinin-conjugated horseradish peroxidase (WGA-HRP) injected unilaterally in the ventral cochlear nucleus (VCN) ipsilateral to the lesion. In this simple transection experiment no regeneration was observed. However, when embryo brainstem tissue was transplanted on the transected site, regeneration occurred in about 30% of the rats examined. These results indicate the occurrence of obvious regeneration in the central auditory pathway, which was previously thought not to be restored once damaged, and they raise hope for future treatment of humans.

Collateral sprouting of central noradrenergic neurons during aging: histochemical and neurochemical studies in intraocular triple transplants.

The sprouting capacity of aged noradrenergic neurons of the brain-stem nucleus locus coeruleus (LC) was examined using intraocular transplants of fetal tissues. Fetal hippocampal tissue (E18) and LC tissue (E15) were transplanted together as a double transplant into the anterior chamber of the eye of young adult Fischer 344 rats. The double transplants were allowed to mature for 14-18 months, after which an additional fetal hippocampal transplant was placed next to the LC graft. The triple transplants were monitored for overall growth and vascularization for an additional 2-6 months. Immunohistochemical examinations showed that both young (2-6 months old) and aged (16-24 months old) hippocampal cografts contained a plexus of thin varicose tyrosine hydroxylase (TH)-immunoreactive fibers extending throughout the grafted hippocampal tissues. However, the aged hippocampal grafts contained a denser uniform plexus of TH-positive fibers compared to the young transplants. Immunohistochemistry with synapsin antibodies demonstrated that both the young and the aged hippocampal transplants contained much higher densities of synaptic elements than the LC grafts. In vivo electrochemical measurements of potassium-evoked overflow of norepinephrine (NE) in the grafts showed that similar amounts of NE overflow were detected in both the young and the aged hippocampal grafts. HPLC-EC measurements of NE levels in the grafts revealed that there were similar amounts of NE in the young and the aged grafts, and the grafts did not contain serotonin or dopamine. In summary, the findings of the present study show that aged LC neurons are capable of undergoing collateral sprouting producing a functional NE neuronal system when introduced to an appropriate young target.

Changes in multiple brain regions underlie species differences in a complex, congenital behavior.

The evolutionary brain modifications that produce any complex, congenital behavioral difference between two species have never been identified. Evolutionary processes may (i) alter a single, "higher" brain area that generates and/or coordinates the diverse motor components of a complex act; (ii) separately change independent, "lower" brain areas that modulate the fine motor control of the individual components; or (iii) modify both types of areas. This study explores the brain localization of a species difference in one such behavior, the crowing of chickens (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Two major subcomponents of the behavioral difference can be independently transferred with interspecies transplantation of separate brain regions, despite the fact that these components, sound and patterned head movement, occur together in a highly integrated fashion. To our knowledge, this is the first experimental demonstration that species differences in a complex behavior are built up from separate changes to distinct cell groups in different parts of the brain and that these cell groups have independent effects on individual behavioral components.

Synapsin I in intraocular hippocampal transplants during maturation and aging: effects of brainstem cografts.

The role of target innervation for maintenance of synaptic proteins in the hippocampal formation during aging was investigated. Fetal CA1 tissue and brainstem tissue containing the nucleus locus coeruleus was dissected from albino rats and grafted sequentially into the anterior chamber of the eye of adult rat recipients. Synapsin protein distribution and levels were evaluated by immunohistochemistry and quantitative immunolabeling in single hippocampal grafts or brainstem-hippocampal double grafts at 6, 12, or 24 mo postgrafting. The synapsin levels in 6-mo-old single hippocampal transplants were significantly lower than those in situ, and remained at these lower levels at 12 and 24 mo. On the contrary, synapsin levels were close to normal in the hippocampal portion of double grafts in the 6- and the 12-mo-group. However, in the 24-mo-old double transplants the levels had declined significantly, approaching levels seen in single hippocampal grafts. The immunoblot results were supported by morphological observations with synapsin antibodies and immunohistochemistry. The present data demonstrate that hippocampal tissue maintained near normal synapsin levels when grafted together with brainstem tissue, as compared to the lower levels seen in single hippocampal grafts. This normalization of synapsin levels was, however, not seen in the aged hippocampal-brainstem double grafts.

Survival and maturation of heterotopic fetal brain stem xenografts after treatment with 2-chlorodeoxyadenosine and cyclosporine A.

Brain stem halves from fetal rabbits were transplanted to the caudate nucleus area of adult rats. The animals were treated postoperatively with cyclosporine A (CsA) and 2-chlorodeoxyadenosine (CdA) for three days, and with CdA alone for the next 13 days. The treatment started at the day of implantation, and in some animals it was repeated starting at day 36 after grafting (at the time when signs of a light inflammatory reaction appeared in some grafts). Grafts survived and matured histologically, and no signs of acute rejection were observed up to the 90th day. In some grafts we recorded phasic neuronal activities similar to the respiratory-related neural activities characteristic for the adult brain stem. Immunosuppressive with CdA and CsA deserves further evaluation in fetal brain grafting.

2-Chloro-2'-deoxyadenosine (2-CdA) combined with cyclosporine A successfully prevents rejection of fetal brain stem allograft in rabbits.

Allografts of brain stem from 20-day-old fetuses to nucleus caudatus of adult rabbits were performed. To prevent graft rejection immunosuppression with 2-CdA and cyclosporine A was transiently induced. Graft survival were assessed by histological and electrophysiological techniques. Both morphological (synaptogenesis, myelinization) and functional (generation of rhythmic neuronal activity) signs of graft maturation were found after nine weeks. The data suggest that transient immunosuppression used is sufficient to induce tolerance to neural graft, and no interference with maturation of implanted fetal tissue occurs.

[A study of transpial migration of implanted serotonergic neurons in rat spinal cord].

Transpial migration of implanted 5-HT neurons from the subarachnoid space into the spinal cord was studied in adult Wistar rats. Embryonic raphe tissue or cell suspension containing 5-HT cells was used as grafts. The implanted 5-HT cells were monitored by 5-HT immunohistochemical method. The results are as follows: (1) 10 d after cutting the spinal cord at lower thoracic level, 5-HT fibers disappeared in the transected spinal cord. (2) Raphe tissue was implanted into the subarachnoid space of the thoracic lumbar segment after the spinal cord was cut. One month later, 5-HT positive cells could be found in the transected spinal cord with fibers extending into both the grey and the white matters. (3) If the raphe cell suspension instead was implanted, a number of 5-HT positive cells appeared in the grey matter near the implanted region and the distribution of these cells in the grey matter was quite consistent with the implanted range of the cell suspension in the subarachnoid space. The 5-HT positive cells which had entered into the spinal cord sent out fibers and reestablished a new fiber network in the grey matter. (4) After implantation, the number of the 5-HT positive fibers in the transected grey matter became more and more sparsely distributed with increasing distance from the cell bodies and the 5-HT positive fibers reappeared in the white matter were much less than that in the grey matter. Present results show that the implanted 5-HT neurons are able to migrate transpially from the subarachnoid space into the spinal cord.

Human fetal xenografts of brainstem tissue containing locus coeruleus neurons: functional and structural studies of intraocular grafts in athymic nude rats.

Fetal human brainstem tissue including the nucleus locus coeruleus was transplanted to the anterior eye chamber of athymic nude rats. Most transplants survived and grew in the anterior chamber of the eye. After 9-15 months, the host animals were anesthetized and electrophysiological or in vivo electrochemical recordings were performed. The brainstem transplants contained spontaneously active neurons with regular single-spike firing patterns. The neurons responded to ipsilateral light stimulation with an increase in firing rate and to the alpha 2-receptor agonist clonidine with significantly decreased firing rates. In vivo electrochemical studies demonstrated reproducible noradrenergic overflow after local application of potassium. Immunohistochemical evaluation of the brainstem transplants showed an abundance of tyrosine hydroxylase-positive neurons and neurites in all transplants and a dense network of neurofilament-, synapsin-, and glial fibrillary acidic protein-positive profiles throughout the grafts. Taken together, the present physiological and histochemical data indicate that it is possible to obtain transplants containing a specific monoaminergic population within the brainstem from human fetal fragments and to maintain these transplants in oculo in athymic nude rats for at least 15 months, during which time noradrenergic neurons develop.

Distribution of the blood-brain barrier in heterotopic brain transplants and its relationship to the lesions of EAE.

The blood-brain barrier (BBB) is recognized as a barrier to the trafficking of molecules and cellular elements into the central nervous system (CNS). Horseradish peroxidase (HRP) exclusion is used as a measure of BBB integrity. The BBB is altered and becomes permeable during the course of experimental allergic encephalomyelitis (EAE). Heterotopic brain transplantation into the anterior eye chamber is a technique for studying genetic influences and the role of individual cell types on the development of EAE. Prior to EAE induction, HRP is excluded from the central portion of the transplant, demonstrating an intact BBB. In contrast, HRP localization is found at the periphery of the transplant, suggesting an incomplete barrier. However, EAE lesions typically occur within the more central regions of the transplant, where the BBB is intact, and not at peripherally located "leaky" areas. This suggests that endothelial cells at intact BBB sites may direct trafficking of lymphocytes (gating) into the CNS during the development of EAE, rather than the passive entry of lymphocytes into the CNS through a leaky BBB.

Explorations of otic transplantation.

Embryonic rat inner ears were transplanted to the anterior chamber of the eyes of adult rats. While considerable development was evident, the structures present were limited to the vestibular division. We hypothesized that this selective survival could be due to the rate of vascularization. To test the effects of graft vascularization we made transplants in which the internal structures were exposed by removing the apex and base of the developing cochlea. The transplants were rapidly vascularized by the iris. Many of the soft labyrinthine structures of the cochlea from 1-day-old donors showed considerable development, including the spiral limbus, basilar membrane, and organ of Corti. To test the possibility that the cochlea requires inductive or trophic support beyond Embryonic Day 15 (E15), we cotransplanted the embryonic inner ear with developing brain stem. In these transplants, we observed improved development of the cochlea, with spiral ganglion cells and an organ of Corti possessing hair cells, Deiter's cells, and pillar cells. To further address the effect of developing CNS tissue on the development of grafted inner ear, we transplanted E15 inner ears to either the cortex or the brain stem of neonatal rats. In these experiments we have seen evidence of both vestibular and cochlear sensory surfaces. In the cochlea, an organ of Corti-like structure can be seen. The possibility of neural connections with the host brain has yet to be investigated.

Age-induced changes in single locus coeruleus brain transplants grown in oculo: an in vivo electrochemical study.

Brain stem tissue from fetal Sprague-Dawley rats containing the nucleus locus coeruleus (LC) was transplanted into the anterior chamber of the eye of young adult host rats and was studied at 4-6 months (young control) or 24-28 months after grafting (old). High-speed in vivo electrochemical measurements were used to characterize the potassium-evoked synaptic overflow of norepinephrine (NE) in both young and aged LC brain grafts. The amplitudes of potassium-evoked NE overflow were attenuated in the aged grafts as compared to the young LC grafts. In addition, the rise times of potassium-evoked responses were longer in the old LC grafts than in the young transplants. In contrast, the NE content of aged LC grafts, as determined by high-performance liquid chromatography coupled with electrochemical detection (HPLC-EC), was only slightly diminished and not significantly different from the NE levels seen in young LC grafts. However, light microscopical evaluation using tyrosine-hydroxylase immunocytochemistry revealed pyknotic cell bodies and fluorescent accumulations in aged locus coeruleus transplants which were indicative of degeneration in these grafts. The present data demonstrate a significant age-related decline in the presynaptic function of NE-containing neurons in intraocular locus coeruleus transplants of Sprague-Dawley rats.

Rearrangement of serotonin-immunoreactive fibers in the denervated rat suprachiasmatic nucleus after transplantation of fetal raphe tissue.

Pieces of fetal midbrain raphe tissue were transplanted into the third ventricle or the ventral hypothalamic region near the suprachiasmatic nucleus (SCN) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. The ability of grafted serotonin neurons to reinnervate the SCN in the host rats was studied by means of immunohistochemistry 1 and 3 months after transplantation. In both the intraventricular and intraparenchymal transplant experiments, reinnervation by outgrowing serotonin fibers was observed in the hypothalamus of host rats at 1 and 3 months after surgery. At both survival periods, there was no abundant arborization of serotonin fibers in the SCN, while the preoptic and periventricular areas of the host rats displayed a pattern of serotonergic innervation resembling that in normal (untreated) rats. It is suggested that within the SCN the regenerating serotonin fibers may be exposed to an inhibitory environment.