Repair strategies for traumatic spinal cord injury, with special emphasis on novel biomaterial-based approaches.

As a part of the central nervous system (CNS), the adult mammalian spinal cord displays only very poor ability for self-repair in response to traumatic lesions, which mostly lead to more or less severe, life-long disability. While even adult CNS neurons have a certain plastic potential, their intrinsic regenerative capacity highly varies among different neuronal populations and in the end, regeneration is almost completely inhibited due to extrinsic factors such as glial scar and cystic cavity formation, excessive and persistent inflammation, presence of various inhibitory molecules, and absence of trophic support and of a growth-supportive extracellular matrix structure. In recent years, a number of experimental animal models have been developed to overcome these obstacles. Since all those studies based on a single approach have yielded only relatively modest functional recovery, it is now consensus that different therapeutic approaches will have to be combined to synergistically overcome the multiple barriers to CNS regeneration, especially in humans. In this review, we particularly emphasize the hope raised by the development of novel, implantable biomaterials that should favor the reconstruction of the damaged nervous tissue, and ultimately allow for functional recovery of sensorimotor functions. Since human spinal cord injury pathology depends on the vertebral level and the severity of the traumatic impact, and since the timing of application of the different therapeutic approaches appears very important, we argue that every case will necessitate individual evaluation, and specific adaptation of therapeutic strategies.

Nanoscale mechanical properties of chitosan hydrogels as revealed by AFM.

In the context of tissue engineering, chitosan hydrogels are attractive biomaterials because they represent a family of natural polymers exhibiting several suitable features (cytocompatibility, bioresorbability, wound healing, bacteriostatic and fungistatic properties, structural similarity with glycosaminoglycans), and tunable mechanical properties. Optimizing the design of these biomaterials requires fine knowledge of its physical characteristics prior to assessment of the cell-biomaterial interactions. In this work, using atomic force microscopy (AFM), we report a characterization of mechanical and topographical properties at the submicron range of chitosan hydrogels, depending on physico-chemical parameters such as their polymer concentration (1.5%, 2.5% and 3.5%), their degree of acetylation (4% and 38.5%), and the conditions of the gelation process. Well-known polyacrylamide gels were used to validate the methodology approach for the determination and analysis of elastic modulus (i.e., Young's modulus) distribution at the gel surface. We present elastic modulus distribution and topographical and stiffness maps for different chitosan hydrogels. For each chitosan hydrogel formulation, AFM analyses reveal a specific asymmetric elastic modulus distribution that constitutes a useful hallmark for chitosan hydrogel characterization. Our results regarding the local mechanical properties and the topography of chitosan hydrogels initiate new possibilities for an interpretation of the behavior of cells in contact with such soft materials.

Cooperation of intrinsic and extrinsic signals in the elaboration of regional identity in the posterior cerebral cortex.

Understanding the compartmentalization of the neocortex (isocortex) of the mammalian brain into functional areas is a challenging problem [1-3] . Unlike pattern formation in the spinal cord and hindbrain, it does not involve the specification of distinct cells types: distinct areas differ in their patterns of connectivity and cytoarchitecture. It has been suggested that signals intrinsic to the neocortical neuroepithelium specify regional fate [3]. Alternatively, spatial patterning might be imposed by extrinsic cues such as thalamocortical projections [4-6]. Recent results highlight the ability of early precursor cells of the telencephalic neuroepithelium to 'remember' their spatial position from times before thalamic innervation [7,8] [9-12]. An influence from the thalamus, however, cannot be ruled out as there is a precise invasion of the correct cortical areas by the corresponding projections [13,14]. Furthermore, cortical neuronal progenitors have been proposed to adopt new connection patterns after transplantation [6,7], as well as when the thalamic input is rerouted [15,16]. Here, we describe the transient expression of the homeobox gene Otx2 in the posterior, prospective visual, neocortex and use it to analyze the establishment of posterior cortical fate. The results suggest that whereas intrinsic cortical information is sufficient to specify regional fate, extrinsic signals from the thalamus are involved in the expansion or maintenance of the population of cells expressing Otx2 but not in regionalization.

Morphofunctional plasticity in the adult hypothalamus induces regulation of polysialic acid-neural cell adhesion molecule through changing activity and expression levels of polysialyltransferases.

Polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression in the adult nervous system is restricted to regions retaining a capacity for morphological plasticity. For the female rat hypothalamoneurohypophysial system (HNS), we have previously shown that lactation induces a dramatic decrease in PSA-NCAM, while leaving the level of total NCAM protein unchanged. Here, we wanted to elucidate the molecular mechanisms leading to a downregulation of PSA, thereby stabilizing newly established synapses and neurohemal contacts that accompany the increased activity of oxytocinergic neurons. First, we show that the overall specific activity of polysialyltransferases present in tissue extracts from supraoptic nuclei decreases by approximately 50% during lactation. So far, two polysialyltransferase enzymes, STX and PST, have been characterized for their capacity to transfer PSA onto NCAM in vitro. Using a competitive RT-PCR on RNA extracts from the HNS, we demonstrate furthermore a significant decrease in the expression levels of both STX and PST mRNAs in lactating versus virgin animals. Interestingly, this downregulation of NCAM polysialylation is not correlated with the post-transcriptional regulation of variable alternative spliced exon splicing, in contrast to neural development. The control of polysialylation via a regulation of both enzyme activity and expression underlines the important role of this post-translational modification of NCAM in morphofunctional plasticity in adult brain.

Stage- and region-specific expression of estrogen receptor alpha isoforms during ontogeny of the pituitary gland.

The expression time course of estrogen receptor alpha (ER alpha) was analyzed by RT-PCR in fetal and newborn rat pituitaries. In addition to the classical ER alpha messenger RNA (mRNA), three shorter transcripts were detected and subsequently cloned. Sequence analysis showed that they corresponded to ER alpha mRNAs lacking exon 3 (which encodes a zinc finger in the DNA-binding domain), exon 4 (which encodes the nuclear localization signal and part of the steroid-binding domain), or both exons 3 and 4. As analyzed by RT-PCR and ribonuclease protection assay, the respective expression levels of the different transcripts varied dramatically during pituitary development; short forms appeared 4 days before full-length ER alpha mRNA. On Western blots from rat pituitaries of different ages, an ER alpha-specific antiserum labeled four protein bands of the expected molecular weights, revealing that all four ER alpha mRNAs are translated in vivo. Immunocytochemistry, using the same antiserum, showed the ER alpha to be present first in the cytosol of intermediate lobe cells (around embryonic day 16). Only 5 days later, nuclear staining became detectable in the anterior lobe. We argue that the observed cytosolic staining will be essentially due to short ER alpha isoforms, which are indeed more abundantly expressed in the intermediate lobe. These data suggest that during pituitary development, the activity of the ER alpha might be specifically regulated by differential splicing of its primary transcript, resulting in a differential subcellular localization of the isoforms.

Homotypic fetal transplants into an experimental model of spinal cord neurodegeneration.

Many neurotransplantation studies have dealt with the ability of solid fetal spinal grafts to develop in the previously traumatized spinal cord of a host. In neurodegenerative spinal diseases, however, motoneuronal death occurs in the absence of a trauma, i.e., in the absence of axotomy of afferent fibers. Lesioning the spinal cord with an excitotoxic agent may provide a useful neurodegenerative model. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension are able to rebuild a neural circuitry. Emphasis is given here to the analysis of the development of transplanted motoneurons and host-graft connectivity. The lesion was made by kainic acid on the right side of the lumbar enlargement 1 week before transplantation. The fetal spinal cords were taken from rat embryos (gestational day E12-13) and transplanted as cell suspensions. Light- and electron-microscopic analysis demonstrated that the excitotoxic lesion extended over the entire spinal segment and was confined primarily to the ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. The lesion was characterized by a lack of neurons, glial proliferation, and sparing of fibers of passage and afferents. Two to fourteen months after surgery, the transplants were generally large, occupying most of the neuron-depleted area. The boundaries between the transplant and host tissue were clearly delineated by the higher cellular density of the graft and the particular cytoarchitecture, i.e., the cell suspension grafts did not display a laminar organization. Among the different neuronal populations within the transplant, one resembled motoneurons: large, typically Nissl-stained and immunoreactive for calcitonin gene-related peptide (CGRP). No grafted neuron, however, extended an axon into the host ventral roots. Monoaminergic afferents from the host were studied using immunostaining for serotonin, noradrenaline, and tyrosine hydroxylase. These afferent fibers, thin and varicose, grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers (entering the graft from the dorsal host-graft interface) penetrated deeply into transplants. The response of cortico- and rubro-spinal afferents to the implantation of fetal tissue was different. After injection of WGA-HRP, a few anterogradely labeled cortical and rubral fibers entered only the most peripheral portion of transplants. In conclusion, our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Restoration of substance P and calcitonin gene-related peptide in dorsal root ganglia and dorsal horn after neonatal sciatic nerve lesion.

Dorsal root ganglion (DRG) neurons decrease their substance P (SP) synthesis after peripheral nerve lesions. Levels in the dorsal horn also decline but return to normal if regeneration is successful. In adults, when regeneration is prevented, recovery of SP in the dorsal horn is slow and incomplete, whereas in newborns, recovery is rapid and complete even though retrograde cell death of DRG neurons is greater than in adults. We have examined the mechanisms that might account for the rapid and complete recovery of SP and calcitonin-gene related peptide (CGRP) in the dorsal horn after peripheral nerve injury in newborns. Peptides were compared in the L4 and L5 DRG and spinal cord segments of normal rats and in rats surviving 6 days to 4 months after sciatic nerve section/ligation within 24 hours of birth. Sciatic nerve section/ligation produced 50% neuron death in L4 and L5 DRGs, but immunocytochemical methods showed that both SP-immunoreactivity (-IR) and CGRP-IR recovered completely in dorsal horn. Radioimmunoassay confirmed that recovery of SP was not an artefact due to shrinkage. beta-Preprotachykinin (PPT)-mRNA hybridization and SP-IR were observed mostly in small neurons; alpha-CGRP-mRNA-hybridized and CGRP-IR neurons were more heterogeneous. The percentage of DRG neurons that contained SP (approximately 25%) or CGRP (approximately 50%) was the same in normal newborn and adult rats. Neither selective cell survival nor change in neuron phenotype was likely to contribute to the recovery seen in the dorsal horn, and DRG neurons ipsilateral to the lesion exhibited the same level of hybridized beta-PPT-mRNA and alpha-CGRP-mRNA as intact DRG neurons. Because neither the constitutive level of expression of the genes nor peptide levels increased above those observed in intact DRG neurons, these mechanisms were also not responsible. Axotomized DRG neurons, however, contributed to recovery. Recovery was also due to sprouting by neurons in intact DRGs rostral and caudal to L4 and L5.

Expression of a phosphorylated isoform of MAP1B is maintained in adult central nervous system areas that retain capacity for structural plasticity.

Microtubule-associated protein IB (MAP1B) is the first MAP to be detected in the developing nervous system, and it becomes markedly down-regulated postnatally. Its expression, particularly that of its phosphorylated isoform, is associated with axonal growth. To determine whether adult central nervous system (CNS) areas that retain immunoreactivity for MAP1B are associated with morphological plasticity, we compared the distribution of a phosphorylated MAP1B isoform (MAP1B-P) to the distribution of total MAP1B protein and MAP1B-mRNA. Although they were present only at very low levels, both protein and message were found ubiquitously in almost all adult CNS neurons. The intensity of staining, however, varied markedly among different regions, with only a few nuclei retaining relatively high levels. MAP1B-P was restricted to axons, whereas total MAP1B was present in cell bodies and processes. Relatively to total MAP1B protein and its mRNA, MAP1B-P levels decreased more dramatically with maturation, and they were detectable in only a few specific areas that underwent structural modifications. These included primary afferents and motor neurons, olfactory tubercles, habenular and raphe projections to interpeduncular nuclei, septum, and the hypothalamus. The distribution pattern of MAP1B-P was compared to that of the embryonic N-CAM rich in polysialic acid (PSA-NCAM). We found that the PSA-NCAM immunostaining was largely overlapped with that of MAP1B-P in the adult CNS. These results suggest that, like PSA-NCAM, MAP1B may be one of the molecules expressed during brain development that also plays a role in structural remodeling in the adult.

Induction of MAP1B phosphorylation in target-deprived afferent fibers after kainic acid lesion in the adult rat.

We have previously shown that the phosphorylated form of microtubule-associated protein 1B (MAP1B-P), which is located in growing axons during development and regeneration, remains detectable in the adult central nervous system only in areas that undergo morphologic plasticity (Nothias et al. [1996] J. Comp. Neurol. 368:317-334). Our objective in the present study was to determine whether lesion-induced axonal remodeling, in the adult rat, is associated with reinduction of MAP1B phosphorylation. MAP1B-P was not detectable in intact adult thalamic ventrobasal complex (VB), although low levels of MAP1B and its mRNA were present. A neuron-depletion of VB by in situ injection of kainic acid was followed by an induction of MAP1B phosphorylation by 24 hours postlesion. MAP1B-P was detected in fibers originating from undamaged neurons that were not located in the lesion, as demonstrated by the absence of hybridized MAP1B-mRNA. Ultrastructural analysis confirmed the exclusive location of MAP1B-P in axons in a proximodistal gradient. MAP1B phosphorylation appeared to be regulated by posttranslational modification of existing protein because the levels of MAP1B-mRNA did not change. The number of MAP1B-P-labeled fibers increased during the first month postlesion and remained high for a long period. Double staining by using axonal tracing with dextran-biotin and tyrosine hydroxylase immunohistochemistry, showed the presence of MAP1B-P in VB afferents from somatosensory relays and the locus coeruleus. This study supports the hypothesis that MAP1B, at a particular state of phosphorylation, is correlated with axonal remodeling in the adult central nervous system (CNS). We suggest that the interaction of MAP1B-P with microtubules allows the modulation of their dynamic properties during periods of increased axonal plasticity.

First month of development of fetal neurons transplanted as a cell suspension into the adult CNS.

It has been demonstrated elsewhere that fetal thalamic tissue, when transplanted as a cell suspension into the excitotoxically neuron-depleted adult somatosensory thalamus, can grow, differentiate, and receive projections from host afferents. In the present study, we used the same paradigm to analyse the transplanted neurons during their morphogenesis, i.e. during the first month after transplantation. Using various anatomical criteria, at the light and electron microscope levels, we compared the development of transplanted neurons with the normal ontogeny of homologous neuronal populations. Confined solely to the mechanically lesioned area during implantation at seven days post-grafting, the transplant increased in size to occupy most of the previously neuron-depleted area by the third week after grafting. The final size of the transplant thus depended upon the size of the lesion. At seven days post-grafting, the neurons were small in size and the cellular density was high. At this immature stage few synaptic contacts were visible and the ultrastructure was characterized by large extracellular spaces. At 10 days post-grafting, the size of the neurons had increased and the cellular density had decreased. Both an extensive dendritic proliferation and a simultaneous active synaptogenesis could also be observed. All these events continued to evolve and during the third week the neuropil progressively acquired more mature ultrastructural characteristics. Synaptic contacts exhibiting characteristics comparable to those observed in the intact thalamus also became more numerous. At 20 days post-grafting, axonal myelination had started, the development of the graft apparently stopped and the various criteria had stabilized. Until that developmental stage, growth of grafted neurons compared to that of normal thalamic ones. At later stages, however, grafted neurons failed to grow larger and did not reach the size of the homologous population in the adult animal. It seems, therefore, that transplants of thalamic fetal neurons can be used as a tool with which to study thalamic neuronal development, within definable limits.

Dissimilar responses of adult thalamic monoaminergic and somatosensory afferent fibers to implantation of thalamic fetal cells.

It is generally accepted that transplanted fetal neurons can, after several weeks to months, establish connections with the host CNS. Host afferent systems seem, however, to show different types of responses to the presence of grafted fetal neurons. The present study is a preliminary step to identify mechanisms involved in the reactions of adult axons to transplanted fetal neurons. The right ventrobasal thalamus of adult rats was depleted of neurons by in-situ injection of kainic acid and cell suspensions from homotopic thalamic embryonic primordia which were injected into the lesioned area. After various post-implantation delays, ranging from five to 30 days, two types of experiments were performed: (i) noradrenaline and serotonin immunohistochemistry with specific antibodies on alternate sections; and (ii) anterograde tracing using wheat germ agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei and the principal sensory trigeminal nucleus. Five days after transplantation, host monoaminergic fibers (either noradrenergic or serotoninergic) had already grown into the transplants. Ingrowing fibers were thin and poorly varicose, exhibiting endings morphologically similar to the growth cones observed during axogenesis. Seven days after grafting, growth cones were no longer visible and monoaminergic fibers exhibited either normal-sized or very large varicosities. Large varicosities progressively decreased in number and, after three weeks, the fibers displayed a normal adult morphology, forming a dense network all over the transplants. In contrast, host somatosensory afferents, labeled by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, did not grow into the transplants. Intermingling of somatosensory afferents and transplanted cells was observed only after 10 days, when grafted neurons extended outside the original transplantation site into the neuron-depleted area containing the somatosensory afferents. The present results demonstrate that adult monoaminergic and somatosensory afferents, when deprived of their usual target, do not react in a similar way to the addition of fetal neurons. It is proposed that adult monaminergic fibers have the ability to regain morphological (and probably functional) immature forms which were considered to be restricted to the period of axogenesis or to lesion-induced regeneration. In contrast, fetal transplants do not seem to induce, by themselves, a similar alteration of genetic expression in adult somatosensory neurons. It has been proposed that "diffuse" and "point-to-point" axonal systems may be differentiated in the CNS on anatomical bases. The present results add to the identification of two different systems by demonstrating that, in the thalamus, they present dissimilar responses to the implantation of fetal cells.

Regulation of the expression and phosphorylation of microtubule-associated protein 1B during regeneration of adult dorsal root ganglion neurons.

Microtubule-associated protein 1B is a major constituent of the neuronal cytoskeleton during the early stages of development. This protein and its phosphorylated isoform, microtubule-associated protein 1B-P, defined by the monoclonal antibody 1B-P [Boyne L. J. et al. (1995) J. Neurosci. Res. 40, 439-450], are present in growing axons and concentrated in the distal end near the growth cone. In most regions of the central nervous system, microtubule-associated protein 1B and microtubule-associated protein 1B-P are developmentally down-regulated. They remain, however, at relatively high levels in the adult peripheral nervous system, where microtubule-associated protein 1B-P is localized exclusively in axons. The aim of this study was to examine the levels of microtubule-associated protein 1B and its phosphorylated isoform during regenerative growth of peripheral axons. Following transection and re-apposition of the sciatic nerve at midthigh, the levels of total microtubule-associated protein 1B, microtubule-associated protein 1B-P and microtubule-associated protein 1B messenger RNA were analysed in dorsal root ganglion neurons and sciatic nerve axons using western blots and RNase protection assays. After the lesion, there was a small decrease in the levels of microtubule-associated protein 1B and its messenger RNA in dorsal root ganglion neurons. The proximal axonal stump showed a similar decrease in the levels of microtubule-associated protein 1B 30days after lesion and returned to normal 60-90days post-lesion. In the distal stump of the sciatic nerve, the levels of microtubule-associated protein 1B increased dramatically and rapidly between three and 14days, but the protein was localized mainly in activated Schwann cells and myelin-like structures, and not in axons [Ma D. et al. (1999) Brain Res. 823, 141-153]. With the regeneration of axons into the distal stump, an intense expression of microtubule-associated protein 1B was observed in these axons. Microtubule-associated protein 1B-P, however, disappeared from the degenerated distal axonal stump as early as three days post-operation, and was absent in the regenerating axons and in Schwann cells between three and 14days. The levels of microtubule-associated protein 1B-P recovered slowly and did not reach the normal levels even after 90days post-operation. In contrast to the response following transection, the levels of microtubule-associated protein 1B and microtubule-associated protein 1B-P were much less affected after nerve crush. We propose that the relatively high levels of microtubule-associated protein 1B and its messenger RNA in adult dorsal root ganglions support peripheral neuron regeneration. The presence of microtubule-associated protein 1B in the regenerating axons suggests that microtubule-associated protein 1B is involved in axonal growth during peripheral nerve regeneration. However, the phosphorylated microtubule-associated protein 1B-P isoform, associated with growing axons during development, is not present in the regenerating axons after transection, presumably because of changes in the activities of kinases and phosphatases associated with the injury. These observations underscore the difference between axonal development and regeneration and the importance of injury-related effects that occur locally.

The expression and distribution of tau proteins and messenger RNA in rat dorsal root ganglion neurons during development and regeneration.

Microtubule-associated proteins contribute to the balance between stability and plasticity of the neuronal cytoskeleton by modulating assembly and disassembly of microtubules. The tau microtubule-associated proteins exist in several isoforms which are developmentally regulated and differentially distributed. Our objective was to characterize the distribution of tau isoforms in developing and mature dorsal root ganglia neurons and during axonal regeneration following sciatic nerve axotomy. Immunocytochemical analysis was carried out using antibodies that recognize all tau isoforms and a novel antibody that specifically recognizes the high molecular weight isoform. The expression of tau is highly regulated during development. At E14, all dorsal root ganglion neurons express only the low molecular weight tau isoforms. These isoforms are still present in all dorsal root ganglion neurons in neonates, whereas high molecular weight tau isoforms are expressed in a subset of dorsal root ganglion neurons. The switch from low to exclusively high molecular weight tau expression begins at E18 and is completed during the first postnatal week. In the adult, high molecular weight tau is restricted to small- and medium-sized dorsal root ganglion neurons; its distribution largely coincides with the population of substance P and calcitonin gene related peptide peptidergic neurons. This differential distribution was observed in the cell body, dorsal roots and sciatic nerve axons. In contrast to the protein, however, the distribution of high molecular weight tau messenger RNA is not restricted; all dorsal root ganglion neurons express similar tau messenger RNA levels. The discrepancy between the distribution of protein and messenger RNA suggests control at the post-transcriptional or translational levels. Sciatic nerve axotomy which is followed by axonal regeneration did not alter the differential distribution of high molecular weight tau immunostaining. We conclude that the distribution and expression of tau isoforms during axonal regeneration in adult does not recapitulate the developmental pattern.

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum--I. Subcortical afferents.

Subcortical afferents to transplants of fetal striatal tissue, implanted into the excitotoxically lesioned striatum of adult recipient rats, were studied with retrograde and anterograde axonal tracers and immunohistochemistry. One week after a striatal ibotenic acid lesion, involving most of the head of the caudate-putamen, a suspension of fetal striatal tissue (embryonic day 14-15) was injected into the lesioned area. In one group of rats, the ibotenic acid lesion was preceded (10 days) by large intrastriatal injections of True Blue, with injection sites matching the area to be lesioned. This was done to retrogradely pre-label the host brain afferents to the area of the striatum later to be lesioned and grafted. At 3 or 6 months post-transplantation, small injections (50 nl) of rhodamine-labelled latex beads were made into the striatal grafts. In animals where the injections were confined to the graft, retrogradely labelled host brain neurons were found in the thalamus, the substantia nigra, amygdala and dorsal raphe nucleus. Double-labelling analysis revealed that the vast majority of the rhodamine bead-labelled neurons also contained True Blue, which indicates that the host afferents to the graft, to a large extent, were derived from the neurons which normally project to the area of the caudate-putamen which was lesioned by the ibotenic acid injection. To further substantiate these observations a second group of lesioned and grafted animals received unilateral wheatgerm agglutinin-horseradish peroxidase injections into the ipsilateral host thalamus at 4 months post-transplantation in order to anterogradely label the host thalamostriatal axons. In a third group of animals serotonin immunocytochemistry was performed in order to detect possible afferents from the raphe nuclei. In contrast to the serotonin-containing fibers, which were fairly evenly distributed throughout the graft tissue, the peroxidase-labelled thalamic afferents were most prominent in the peripheral zones of the grafts and they were densely aggregated at the graft-host interface. The combined results provide evidence that the intrastriatal grafts receive afferents from the host substantia nigra, thalamus, amygdala and dorsal raphe nucleus, but with different distributions. The afferents from the substantia nigra, amygdala and raphe nuclei seem to distribute throughout the grafted tissue, although they are most dense in the peripheral parts, whereas the thalamic afferents are largely confined to the peripheral areas of the transplants and to the graft-host interface.

Glutamic acid decarboxylase gene expression in thalamic reticular neurons transplanted as a cell suspension in the adult thalamus.

The goal of the present study was to determine whether alterations in neuronal morphology and connections in thalamic grafts were accompanied by changes in the expression of mRNA encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of GABA, the normal neurotransmitter of neurons of the thalamic reticular nucleus. Cell suspensions of rat fetal tissue containing both thalamic reticular nucleus and ventrobasal primordia were transplanted into the excitotoxically lesioned somatosensory thalamus of adult rats. Levels of messenger RNA (mRNA) encoding GAD (Mr 67,000; GAD67) were measured 7 days to 4 months following transplantation via quantitative in situ hybridization with 35S-radiolabeled antisense RNAs. Expression of GAD67 mRNA in the thalamic reticular nucleus was analyzed in parallel in rat pups between 0 and 30 days postnatally, and in adult animals. As already observed with immunohistochemistry, transplanted neurons of the thalamic reticular nucleus did not group in specific clusters but rather mingled with unlabeled (putatively ventrobasal) neurons. Levels of labelling for GAD67 mRNA per neuron increased over time and reached adult levels during the third week post-grafting, i.e. 2 weeks after the theoretical birthdate of the neurons (grafted at embryonic days 15-16). Similar values were observed and a plateau was reached at similar time points during normal ontogeny. The results suggest that, in contrast to morphology and size of the neuronal cell bodies, gene expression of GAD67 develops normally despite the ectopic location of neurons of the thalamic reticular nucleus in the somatosensory thalamus, the abnormal connectivity and the lack of segregation from non-GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal neural transplants into an area of neurodegeneration in the spinal cord of the adult rat.

Lesioning the spinal cord with an excitotoxic agent provides a model of neuronal degeneration while sparing afferent axons. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension were able to rebuild a neural circuitry in the neuron-depleted adult cord. Fetal spinal cords, taken from rat embryos (gestational day E12-13), were transplanted as cell suspensions into an area of the lumbar cord previously depleted of neurons using kainic acid. The excitotoxic lesion extended over ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. During the first month after injection, the damaged cord was characterized by proliferation and recruitment of various glial cell and Schwann cell populations. First to appear were activated microglia/macrophages and next reactive astrocytes which entered the lesion from its borders with the intact tissue. Schwann cells also ensheathed central axons. Differential sensitivity of various afferents to loss of postsynaptic target neurons was observed: rubrospinal and corticospinal afferents decreased in density while no conspicuous changes were observed for immunostained CGRP-containing or monoaminergic fibers. Two to fourteen months after surgery, transplants occupied most of the neuron-depleted area. The grafts did not display a laminar organization. Monoaminergic afferents grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers entering in the dorsal roots penetrated deeply into transplants. In contrast, cortico- and rubrospinal afferents entered only the most peripheral portion of transplants. Our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord. Host-to-graft connections can be formed, although their spatial extent in the transplants may depend upon features of the afferent fiber systems.

Somatotopic reciprocal connections between the somatosensory cortex and the thalamic Po nucleus in the rat.

Retrogradely labeled neurons are observed in the posterior group of the thalamus (Po) after injection of wheatgerm agglutinin-horseradish peroxidase in the rat somatosensory cortex. These neurons are organized in rods elongated rostrocaudally, defining a clear somatotopic map. Injections of tritiated leucine in the somatosensory cortex indicate that these somatotopically organized connections are reciprocal. Injections of tritiated leucine in the dorsal column nuclei label afferent fibers in a small area dorsal to Po but not in the core of the nucleus. Po does not receive direct projections of ascending somatosensory afferents. It is hypothesized that this thalamic area participates in a thalamo-cortico-thalamic loop.

Rapid growth of host afferents into fetal thalamic transplants.

Fetal cell suspension grafts grow and differentiate when implanted into adult rat CNS areas previously neuron-depleted using an excitotoxin. There is some controversy in the literature concerning the timetable of establishment and possible extent of host-graft connections in these experimental conditions. The present study was undertaken to analyze the development of adult host monoaminergic afferents into a transplant formed by fetal thalamic neurons in the previously excitotoxically lesioned thalamus. It is demonstrated that both norepinephrin- and serotonin-immunoreactive fibers are present in the transplant as soon as 8 days after grafting. At those times, immunoreactive fibers exhibit morphological characteristics typically associated with immature stages. After longer survival time, up to 4 weeks after grafting, immunoreactive fibers are numerous in the transplant and exhibit morphological features comparable to those observed in the adult thalamus. These results demonstrate the rapid ingrowth of some fiber systems of the adult host into the transplant and suggest that grafted fetal cells can be functionally integrated into the host circuitry as soon as a few weeks after grafting.

Morphological alteration of thalamic afferents in the excitotoxically lesioned striatum.

Excitotoxic lesions of the neostriatum cause anatomical and biochemical changes resembling those occurring in Huntington's disease. One major characteristic of these lesions is that they acutely spare axons of passage and afferent fibers. However, evidence is accumulating that afferent axonal systems decrease their fiber density in the long-term excitotoxic lesion. Ultrastructural changes of neuron-deprived terminals may also occur. A parallel study considering changes in afferent fibers to the excitotoxically lesioned thalamus showed that, a few weeks after neuron-depletion, specific 'point-to-point' systems formed regenerating axonal growth cone-like structures. The present study used the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to determine whether specific thalamostriatal afferents form the same kind of regenerating structures following excitotoxic lesion of their target neurons. Thalamostriatal afferents decreased in density over months after lesion, but some were still labeled as long as 4 months after ibotenic acid injection. Remaining afferents formed axonal growth cone-like structures, identified at both light and electron microscopic levels, similar to those observed in the lesioned thalamus. These results demonstrate that in the striatum as in the thalamus, neuron depletion is followed by a long-term alteration of the morphology of some afferent fibers which form regenerating growth cone-like structures. These results are discussed with regard to the possible functional integration of fetal neurons transplanted into previously excitotoxically lesioned areas.

Vascularization of fetal cell suspension grafts in the excitotoxically lesioned adult rat thalamus.

Several studies have considered the establishment of vascularization in intracerebral solid transplants of neural tissue. The widely supported interpretation of the results is that the vascular network of the solid grafts is already present before implantation into the host brain. The situation is different when dissociated fetal tissue is transplanted as a cell suspension because in these conditions the fetal vascular network is disrupted. The present study has, therefore, been undertaken to follow the angiogenesis in a transplant of dissociated fetal cells implanted into the excitotoxically neuron-depleted thalamus. The vascular network is compared to that observed in the intact and in the lesioned thalamus both in terms of morphology of the capillaries and of the function of the blood-brain barrier (BBB). In the transplant, capillaries, stained by Indian ink, are very few in number and have very fine calibers during the first 20 days after grafting. Some structures can be identified as immature blood vessels at the electron microscopic level. The blood vessels are progressively more numerous in the graft and they demonstrate mature ultrastructural features 2 months after grafting. Last, there is no leakage of the BBB for peroxidase. The vascularization seems to follow a pattern of maturation comparable to that described during development in the literature. In contrast, in the lesioned area, there is a reactive angiogenesis: 10 days after the excitotoxic injection (shortest time studied), there are many wide caliber vessels with expanded perivascular spaces engorged with mesodermal cells. A microvascularization also develops transiently during the first two months. Capillaries are abnormal from the functional point of view, since there is a leakage of the BBB to macromolecules. The use of an experimental model in which transplant had to grow in a lesioned area permits to determine two types of vascularization: an apparently normal developmental timetable, normal morphological and functional characteristics, in the transplant; a reactive angiogenesis, in the lesioned area.