The use of stem cells in regenerative medicine for Parkinson's and Huntington's Diseases.

Cell transplantation has been proposed as a means of replacing specific cell populations lost through neurodegenerative processes such as that seen in Parkinson's or Huntington's diseases. Improvement of the clinical symptoms has been observed in a number of Parkinson and Huntington's patients transplanted with freshly isolated fetal brain tissue but such restorative approach is greatly hampered by logistic and ethical concerns relative to the use of fetal tissue, in addition to potential side effects that remain to be controlled. In this context, stem cells that are capable of self-renewal and can differentiate into neurons, have received a great deal of interest, as demonstrated by the numerous studies based on the transplantation of neural stem/progenitor cells, embryonic stem cells or mesenchymal stem cells into animal models of Parkinson's or Huntington's diseases. More recently, the induction of pluripotent stem cells from somatic adult cells has raised a new hope for the treatment of neurodegenerative diseases. In the present article, we review the main experimental approaches to assess the efficiency of cell-based therapy for Parkinson's or Huntington's diseases, and discuss the recent advances in using stem cells to replace lost dopaminergic mesencephalic or striatal neurons. Characteristics of the different stem cells are extensively examined with a special attention to their ability of producing neurotrophic or immunosuppressive factors, as these may provide a favourable environment for brain tissue repair and long-term survival of transplanted cells in the central nervous system. Thus, stem cell therapy can be a valuable tool in regenerative medicine.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Behavioral and morphological consequences of primary astrocytes transplanted into the rat cortex immediately after nucleus basalis ibotenic lesion.

Adult male rats received transplants of dissociated 30-day old cultured cortical astrocytes into the ipsilateral frontal and parietal cortex immediately after unilateral ibotenic acid lesion of the NBM or after sham injury. We hypothesized that transplants of astrocytes into the acetylcholine-deprived cortex might provide trophic support to terminals arising from damaged NBM neurons. Twenty four hours after transplantation and every other day for 11 days post surgery, the animals were tested for locomotion and habituation in an open field. NBM lesion reduced vertical movements only as compared to no lesion and no transplant counterparts. Nine days after surgery rats with NBM lesion and astrocyte-transplants into the cortex were as impaired in the acquisition of a passive avoidance (PA) task as untreated counterparts. Animals with no lesions and transplants into the cortex also had significant PA acquisition deficits. All rats with ibotenic lesion were significantly impaired on PA retention as compared to rats with no lesions. Astrocyte-transplants survived up to 2 months after cortical implantation but these transplants produced severe laminar disruption and gliosis. This effect was greater in rats with NBM lesion than in intact animals with transplants into the cortex. These data show that astrocyte-transplants do not promote functional recovery after NBM lesion and suggest an immune rejection of the astrocyte transplants by the host brain.

Macrophages enhance muscle satellite cell proliferation and delay their differentiation.

This study investigated the effect of macrophages on in vitro satellite cell myogenesis in the turkey and mouse. Macrophages are considered to act as scavengers of tissue debris during the muscle degeneration-regeneration process. The number of dividing cells and of myoblasts expressing the myogenic regulatory factor MyoD indicated that macrophages enhanced satellite cell proliferation in both species. This was confirmed by observations with cultures treated for bromodeoxyuridine (BrdU) incorporation. In mouse and turkey macrophage-satellite cell cocultures, the number of differentiated myoblasts, the frequency of myogenin-positive cells, and the expression of developmental myosin isoforms were reduced as compared with control cultures, indicating that macrophages delayed satellite cell differentiation. The possibility that macrophages facilitate muscle fiber reconstitution by enhancing satellite cell proliferation should be taken into consideration in designing future strategies of satellite cell transplantation as a treatment for muscular dystrophies.

Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant.

The transplantation of satellite cells may constitute a strategy for rebuilding muscle fibres in inherited myopathies. However, its development requires a great understanding of the role of environmental signals in the regenerative process. It is therefore essential to identify the key events triggering and controlling this process in vivo. We investigated whether macrophages play a key role in the course of the regenerative process using skeletal muscle transplants from transgenic pHuDes-nls-LacZ mice. Before grafting, transplants were conditioned with macrophage inflammatory protein 1-beta (MIP 1-beta; stimulating the macrophages infiltration or vascular endothelial growth factor (VEGF) stimulating angiogenesis). Treatment of transplants with MIP 1-beta and VEGF both accelerated and augmented monocyte-macrophage infiltration and satellite cell differentiation and/or proliferation, as compared to controls. In addition, VEGF treatment enhanced the number of newly formed myotubes. When a complete depletion of host monocyte-macrophages was experimentally induced, no regeneration occurred in transplants. Our data suggest that the presence of blood borne macrophages is required for triggering the earliest events of skeletal muscle regeneration. The understanding of macrophage behaviour after muscle injury should allow us to develop future strategies of satellite cell transplantation as a treatment for muscular dystrophies.

MyoD, myogenin, and desmin-nls-lacZ transgene emphasize the distinct patterns of satellite cell activation in growth and regeneration.

Although satellite cell differentiation is involved in postnatal myogenesis from growth to posttrauma regeneration, the early stages of this process remain unclear. This study investigated pHuDes-nls-lacZ transgene activity, as revealed by X-gal staining and the accumulation of MyoD, myogenin, endogenous desmin, and myosin, in order to determine whether satellite cells share the same activation program during growth and regeneration. After birth, skeletal myonuclei in which myogenin expression was limited were briefly characterized by transgene activity. Satellite cells were only evidenced by MyoD and slow myosin accumulation, but failed to initiate transgene expression. After freeze trauma, satellite cell activation led to MyoD, myogenin, and desmin expression. Subsequently, when myosin expression occurred, transgene activation was apparent in regenerating structures, with more intense X-gal staining in mononucleated cells than regenerating myotubes. After the second week posttrauma, only desmin and myogenin expression were maintained in regenerating structures. In culture, the behavior of satellite cells showed that desmin expression was committed before transgene activation occurred, i.e., concurrently with MyoD, myogenin, myosin expression, and the first fusion events. Quantitative analysis confirmed the discrepancy between endogenous desmin and transgene expression and demonstrated the close correlation between transgene activation and the fusion index. Our results strongly suggest that satellite cells promote distinct pathways of myogenic response during growth and regeneration.

Astrocytes grafted into rat nucleus basalis magnocellularis immediately after ibotenic acid injection fail to survive and have no effect on functional recovery.

In order to determine if the "trophic" properties of astrocytes makes them appropriate for use as a therapeutic agent to excitotoxic brain damage, adult male rats received grafts of cultured cerebral cortical astrocytes into the NBM immediately after infusion of ibotenic acid into the same structure. Twenty four hours after grafting and every other day for 11 days post surgery, the animals were tested for locomotor activity and habituation in an open field. Animals with NBM lesions had significantly reduced rearing activity as compared to counterparts with no lesions. Nine days after surgery, rats with NBM lesions and astrocyte grafts were as impaired in the acquisition of passive avoidance (PA) as their untreated counterparts. All animals with ibotenic lesions were impaired on PA retention compared to rats with no lesions. There was no difference between animals that had received grafts and those that had not. Fourteen days after grafting, all brains were processed for Nissl stain, acetylcholinesterase (AChE) histochemistry, GFAP immunocytochemistry, and bisbenzamide fluorescent microscopy. Decreases in the number of neurons in the NBM as well as decreases in the density of AChE staining in the ipsilateral cortex (the area of innervation of the NBM cholinergic neurons) was evident in all animals with NBM lesions. In addition, a large number of host reactive astrocytes were seen within the NBM, its vicinity, and in the ipsilateral neocortex. Grafted astrocytes survived and integrated into the host tissue when they were grafted into the brain of intact animals but no living grafted astrocytes were found in animals injected with ibotenate. In this latter case, two weeks after grafting, instead of surviving astrocytes only fluorescent tissue 'masses' were seen in the NBM, surrounded by a cavity. Grafted astrocytes did not have any effect on the extension of the lesion caused by ibotenic acid infusion. These results suggest that the concentration of ibotenic acid used to injure the NBM killed not only the host cholinergic neurons but also the grafted astrocytes. The failure of astrocytes to ameliorate the behavioral deficits caused by ibotenic acid lesions of the NBM may be due to the ibotenic acid creating a lethal environment for the grafted and freshly dissociated, cultured astrocytes.

Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants.

The purpose of this study was to investigate the initiation and time course of the regeneration process in fragments of skeletal muscle transplants as a function of muscle tissue age at implantation. The appearance of desmin occurs at the very beginning of myogenesis. The transgenic desmin nls lacZ mice used in the study bear a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase. The desmin lacZ transgene labels muscle cells in which the desmin synthesis programme has commenced. We implanted pectoralis muscle fragments from fetal transgenic embryos and mature and old transgenic mice into mature non-transgenic mice. Early events of myogenesis occurring during regeneration started sooner in transplants from 4-month-old (day 3 post-implantation) muscle than in those from 24-month-old (day 5-6 post-implantation) muscle, and they lasted longer in those from young (day 17 post-implantation) than in those from old (day 14 post-implantation) muscle fragments. In adult muscle, transgene activation proceeded from the periphery toward the centre of the transplant. In transplants from fetal 18-day-old pectoralis, myotubes with transgene activity were observed from day 1 to day 19. Desmin immunoreactivity, which appeared about one day after transgene activation, was followed by myosin expression. In adult transplants, the continuity of laminin labelling was disrupted around degenerative fibres, illustrating alteration of the extracellular matrix. Our data suggest that satellite cells from old muscle tissue have lower proliferative capacity and/or less access to trophic substances released by the host (damaged fibres, vascularization) than those from fetal or young adult muscle.

Desmin-lacZ transgene, a marker of regenerating skeletal muscle.

Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which encoded for the enzyme beta-galactosidase were used. In the muscle cell, a blue nuclear product appearing in the presence of the X-gal substrate for the enzyme provided evidence of the expression of the desmin gene. However, no transgene expression was observed 2 weeks postnatal in skeletal muscles, even though endogenous desmin was present. In order to investigate the regulatory mechanisms of the desmin gene during regeneration, adult pectoralis fragments (without expression of the desmin transgene) from transgenic mice were implanted into the tibialis anterior of 4 day or 6 week old Swiss mice. Adult pectoralis transplants reexpressed the transgene from day 4 to 10 after implantation. In addition, lesions were performed in adult transgenic pectoralis and transgenic expression in injured muscles was observed 2 days later. This new transgenic mouse is a powerful tool for the study of the various steps of skeletal muscle regeneration.

Effects of fetal forebrain transplants in ibotenic-injured nucleus basalis: an anatomical investigation.

Survival of fetal basal forebrain transplant (TP) into ibotenic-injured nucleus basalis of rats was examined after a delay lesion and TP (1 or 2 weeks) and a delay between harvest and TP (1-4.5 hours). Optimal TP survival occurred for TP made 2 weeks postlesion and less than 2 hours after harvesting. In these cases large, healthy TP-neurons displayed robust cytochrome oxidase (CO) activity and sent cholinergic processes throughout the TP and occasionally into host tissue. A mild astrocytic reaction was observed within the TP and at the host-TP interface. Surviving TPs increased choline acetyltransferase innervation and CO activity within the ipsilateral frontoparietal cortex. Therefore data suggest that fetal cholinergic TPs into the damaged NBM reduced neuronal degeneration within the NBM and stimulated remaining neurons spared by the lesion.

Comparison of GM1 ganglioside, AGF2, and D-amphetamine as treatments for spatial reversal and place learning deficits following lesions of the neostriatum.

These experiments tested the effectiveness of parenterally administered gangliosides and amphetamine as treatments for spatial learning deficits caused by bilateral lesions of the neostriatum. In Expt. 1, rats were tested postsurgically for 30 days on a shock-avoidance, spatial reversal task. Treatments of gangliosides (GM1 at 30 mg/kg, and AGF2 at 20 mg/kg and 30 mg/kg) and D-amphetamine (2 mg/kg) significantly decreased lesion-induced learning deficits on this task, while treatments of 10 mg/kg AGF2 and the combination of GM1 (30 mg/kg) and D-amphetamine (2 mg/kg) were ineffective. In Expt. 2, rats were given bilateral neostriatal lesions and treated with GM1 (30 mg/kg), AGF2 (20 mg/kg) or D-amphetamine (2 mg/kg) and tested postsurgically for 5 days on a place learning task in the Morris water maze. Only the GM1-treated rats showed a reduction in lesion-induced place learning deficits on this task. Since in both experiments, cell counts near the area of the lesion revealed no differences among any of the brain-damaged groups, it was suggested that the treatments exert their behavioral effects by biochemically activating spared neurons, independent of any ultimate effects they may have on neuronal survival.

Unilateral cortical contusion injury in the rat: vascular disruption and temporal development of cortical necrosis.

Cerebrovascular disruption and cortical pathology resulting from either moderate (M-TBI) or severe (S-TBI) traumatic brain injury produced by a pneumatically-driven cortical contusion device were assessed in adult male rats sacrificed at 6 and 24 h or 8 and 30 days after injury to the right sensorimotor cortex. Epidural, subdural, subarachnoid, petechial (cortex and corpus callosum), and/or intraventricular hemorrhage was present in all animals, more extensively and severely following S-TBI. At 6 or 24 h after TBI, acidophilic (acid fuchsin-positive) neurons were numerous and widespread (S-TBI > M-TBI) in the ipsilateral contused cortex. By 8 days few acidophilic neurons were present in peri-impact regions of the ipsilateral neocortex, and none were detected in cortex 30 days postinjury. Both M-TBI and S-TBI groups had enlarged ipsilateral cortical volumes (edema) at 6 and 24 h post-contusion. Eight and 30 days after injury the mean volume of cortical necrosis was significantly larger in S-TBI than in M-TBI rats, and cortical necrosis in both TBI conditions increased between 8 to 30 days postinjury. These results indicate that this pneumatically-driven contusion device produces reliable and consistent primary and secondary cortical histopathology, the extent of which is related to the severity of initial injury.

GM1 ganglioside effects on astroglial response in the rat nucleus basalis magnocellularis and its cortical projection areas after electrolytic or ibotenic lesions.

We examined the effects of chronic GM1 ganglioside injections on the astroglial response to bilateral electrolytic or ibotenic acid lesions in the nucleus basalis magnocellularis (NBM) within the NBM and in three cortical projection areas of NBM neurons. Glial fibrillary acidic protein (GFAP) immunohistochemistry was used to visualize the reactive astrocytes. Twenty-six days after injury, extensive astrogliosis was observed within the NBM after both types of lesions. An increased number of GFAP-positive cells were found in the cortex of saline-treated rats following electrolytic but not ibotenic lesions. We suggest that the loss of fibers of passage within the lesion area may account for the difference in cortical gliosis following the two types of damage. Although 17 days of GM1 injections did not affect astrocyte morphology within the NBM, ganglioside treatment reduced the number of GFAP-positive cells after electrolytic but not after ibotenic lesions. Within the cortex, a decrease in GFAP immunoreactivity, size, and number of astrocytes was only observed after electrolytic lesion. These data indicate that a decrease in the astroglial response to injury is the result of an interaction between the type of injury (electrolytic lesion) and chronic GM1 treatment.

Differential effects of chronic ethanol consumptions or thiamine deficiency on spatial working memory in Balb/c mice: a behavioral and neuroanatomical study.

This study was aimed to compare the effects of either a thiamine deficiency or a chronic ethanol consumption on memory and on neuronal density within the median mammillary nucleus. Results showed that alcohol-treated (48 weeks) mice exhibited a behavioral impairment in a sequential alternation task characterized by a progressive decay of alternation rates as a function of the number of trials; such a deficit was not observed in controls and thiamine-deficient subjects. A quantitative analysis using histological sections showed an important reduction of neuronal density within the mammillary bodies following the alcohol treatment but not following thiamine deficiency.

Functional recovery following transplants of embryonic brain tissue in rats with lesions of visual, frontal and motor cortex: problems and prospects for future research.

In animals, fetal brain tissue grafts into damaged adult host brain reduce some of the functional deficits caused by brain lesions. Although neurons from transplants survive and develop reciprocal connections with host brain tissue, such connections are generally not enough to replace damaged fibers completely and support behavioral recovery observed. Moreover, grafts never exhibit a normal morphological appearance as compared to adult tissue, but some metabolic activity is occasionally detected within the transplant. Release and/or diffusion of trophic substances from the transplant, in addition to those from the damage host brain, may partially restore neuronal and behavioral functions especially after lesions of the visual cortex. In this case, it can be hypothesized that fetal transplants serve as "living mini-pumps". In addition, there is evidence that the combination of trophic substances (e.g. GM1 ganglioside) and fetal brain transplants may provide a better opportunity for recovery than either treatment given by itself.

Modifications in number and morphology of dendritic spines resulting from chronic ethanol consumption and withdrawal: a Golgi study in the mouse anterior and posterior hippocampus.

Using Golgi-impregnated mice brains, the effects of 2.5, 6.5, and 9.5 months of chronic ethanol consumption were investigated with regard to morphology and number of dendritic spines of CA1 hippocampal pyramidal neurons. The posterior part of the hippocampus was more sensitive to the effects of ethanol consumption than the anterior one. In the posterior part of the structure, the number of dendritic spines was reduced by 25 and 33% after 6.5 and 9.5 months of treatment, respectively, as compared to age-matched controls. Moreover, the remaining spines appeared shorter than normal. After 9 months of ethanol treatment followed by 0.5, 1, and 2 months of withdrawal, the number of dendritic spines was reduced by 24, 19, and 7.5% in the posterior hippocampus, respectively. In the anterior part of the hippocampus, a significant loss of dendritic spines (-20.5%) was observed only after 9.5 months of ethanol consumption. After 1 month of withdrawal, both number and morphology of dendritic spines appeared normal in the anterior hippocampus. These results demonstrate that chronic alcohol consumption leads to morphological alterations and loss of dendritic spines in the hippocampus. However, both dendritic spine number and morphology progressively return to normal values after 2 months of withdrawal. This phenomenon is another example of neuronal plasticity in adult animal brain.

First visualization of dopaminergic neurons with a monoclonal antibody to dopamine: a light and electron microscopic study.

A monoclonal antibody recently synthesized against dopamine (DA) was tested in rat and mouse brain sections after further treatment by PAP immunocytochemistry at the light and electron microscopic levels. Distribution of DA-immunoreactive cell bodies was examined in the substantia nigra (sn), the ventral tegmental area (vta), and the raphe nuclei. DA-immunoreactive fibers were investigated in two DA projection systems, the striatum and the septum. Many dopaminergic cell bodies were found in the sn and the vta. Some scattered DA neurons were encountered in the pars reticulata of the sn. The dorsal raphe and linearis raphe nuclei displayed sparse immunoreactive neurons and a dense plexus of DA fibers. Immunoreactive fibers were observed in the entire striatum, more dense in the ventral part. In the septum, immunonegative neurons were outlined by thin DA fibers in synaptic contact with their somata or dendrites. According to our observations, this DA monoclonal antibody seems to be a selective and sensitive tool for studying the dopaminergic neuronal circuitry at both histological and ultrastructural level.

Build-up and release from proactive interference during chronic ethanol consumption in mice: a behavioral and neuroanatomical study.

Male mice of the BALB/c strain were given a solution of 15% ethanol as their only source of fluid during either 24 or 48 weeks. They were submitted to a sequential alternation (SA) task in a T-maze (6 successive trials). It was found that 48 but not 24 weeks of alcohol administration lead to a deficit as compared to pair-fed or tap-water controls. Whereas experimental mice performed as well as controls on the first 3 choices, they exhibited a gradual decrease in the SA rate on subsequent trials. We suggest that this deficit might result from an exaggerated vulnerability to proactive interference (PI). In order to further test this hypothesis, a second experiment investigated whether a between-trials variation of context of the maze would increase performance. It was found that the SA rate improved as soon as the variation was provided (5th trial). We suggest that the deficit of experimental mice results from an impairment of retrieval processes. A neuroanatomical study was conducted to quantify cell losses resulting from 8, 24 or 48 weeks of ethanol treatment in the mammillary bodies (MM) or the hippocampus (HPC). At the time of appearance of the deficit, MM exhibited a -32% cellular loss, whereas this was only -18% in the HPC. This result emphasises the importance of MM lesion in memory deficits resulting from long-term alcohol consumption.

Immunocytochemical approach of GABAergic innervation of the mouse spinal cord using antibodies to GABA.

The distribution of gamma-amino-butyric acid containing neurons in the Mouse spinal cord has been studied at both the light and electron microscope levels using antibodies against GABA and revelation by the Fab-peroxidase technique. At the light microscope level immunoreactive profiles of perikarya and neuronal processes were particularly abundant in the superficial laminae (I-IV) of the dorsal horn. Scattered soma profiles were found in the other layers and more particularly in the lamina X where Liquor contacting immuno-reactive neurons could be detected. GABAergic cell bodies were very sparse in the ventral horn. Electron microscopic observations confirmed the light microscope results: terminals constituted synaptic symmetrical contacts that provide a morphological basis for inhibition in the dorsal horn and for post-synaptic inhibition of motoneurons in the ventral horn.

Neuroanatomical effects of chronic ethanol consumption on dorsomedial and anterior thalamic nuclei and on substantia innominata in mice.

Quantitative analysis, using histological sections, showed that chronic ethanol consumption for 7 months produced a weak but significant cellular loss in the dorsomedial thalamic nucleus, anterior thalamus and substantia innominata in the mouse. These results are in agreement with patterns of neuroanatomical damage observed in human alcoholics.