Sonic Hedgehog and Triiodothyronine Pathway Interact in Mouse Embryonic Neural Stem Cells.

Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.

Task Force Paper On Cerebellar Transplantation: Are We Ready to Treat Cerebellar Disorders with Cell Therapy?

Restoration of damaged central nervous system structures, functional recovery, and prevention of neuronal loss during neurodegenerative diseases are major objectives in cerebellar research. The highly organized anatomical structure of the cerebellum with numerous inputs/outputs, the complexity of cerebellar functions, and the large spectrum of cerebellar ataxias render therapies of cerebellar disorders highly challenging. There are currently several therapeutic approaches including motor rehabilitation, neuroprotective drugs, non-invasive cerebellar stimulation, molecularly based therapy targeting pathogenesis of the disease, and neurotransplantation. We discuss the goals and possible beneficial mechanisms of transplantation therapy for cerebellar damage and its limitations and factors determining outcome.

Timing correlations between cerebellar interpositus neuronal firing and classically conditioned eyelid responses in wild-type and Lurcher mice.

Classical eyeblink conditioning is an experimental model widely used for the study of the neuronal mechanisms underlying the acquisition of new motor and cognitive skills. There are two principal interpretations of the role of the cerebellum in the learning of eyelid conditioned responses (CRs). One considers that the cerebellum is the place where this learning is acquired and stored, while the second suggests that the cerebellum is mostly involved in the proper performance of acquired CRs, implying that there must be other brain areas involved in the learning process. We checked the timing of cerebellar interpositus nucleus (IPN) neurons' firing rate with eyelid CRs in both wild-type (WT) and Lurcher (a model of cerebellar cortex degeneration) mice. We used delay and trace conditioning paradigms. WT mice presented a better execution for delay vs. trace conditioning and also for these two paradigms than did Lurcher mice. IPN neurons were activated during CRs following the activation of the orbicularis oculi muscle. Firing patterns of IPN neurons were altered in Lurcher mice. In conclusion, the cerebellum seems to be mostly related with the performance of conditioned responses, rather than with their acquisition.

Cerebellum-from J. E. Purkyne up to Contemporary Research.

Jan. Evangelista Purkyne, the most famous among Czech physiologists, was the first who identified and described the largest nerve cells in the cerebellum. The most distinguished researchers of the nervous system then recommended naming these neurons Purkinje cells in his honor. Through experiments by Purkinje and his followers, the function of the cerebellum was properly attributed to the precision of motor movements and skills. This traditional concept was valid until early 1990s, when it was readjusted and replenished with new and important findings. It was discovered that the cerebellar cortex contains more neurons than the cerebral cortex and shortly thereafter was gradually revealed that such enormous numbers of neural cells are not without impact on brain functions. It was shown that the cerebellum, in addition to its traditional role, also participates in higher nervous activity. These new findings were obtained thanks to the introduction of modern methods of examination into the clinical praxis, and experimental procedures using animal models of cerebellar disorders described in this work.

Mutation-related differences in exploratory, spatial, and depressive-like behavior in pcd and Lurcher cerebellar mutant mice.

The cerebellum is not only essential for motor coordination but is also involved in cognitive and affective processes. These functions of the cerebellum and mechanisms of their disorders in cerebellar injury are not completely understood. There is a wide spectrum of cerebellar mutant mice which are used as models of hereditary cerebellar degenerations. Nevertheless, they differ in pathogenesis of manifestation of the particular mutation and also in the strain background. The aim of this work was to compare spatial navigation, learning, and memory in pcd and Lurcher mice, two of the most frequently used cerebellar mutants. The mice were tested in the open field for exploration behavior, in the Morris water maze with visible as well as reversal hidden platform tasks and in the forced swimming test for motivation assessment. Lurcher mice showed different space exploration activity in the open field and a lower tendency to depressive-like behavior in the forced swimming test compared with pcd mice. Severe deficit of spatial navigation was shown in both cerebellar mutants. However, the overall performance of Lurcher mice was better than that of pcd mutants. Lurcher mice showed the ability of visual guidance despite difficulties with the direct swim toward a goal. In the probe trial test, Lurcher mice preferred the visible platform rather than the more recent localization of the hidden goal.

Transplantation of Embryonic Cerebellar Grafts Improves Gait Parameters in Ataxic Lurcher Mice.

Hereditary cerebellar ataxias are severe diseases for which therapy is currently not sufficiently effective. One of the possible therapeutic approaches could be neurotransplantation. Lurcher mutant mice are a natural model of olivocerebellar degeneration representing a tool to investigate its pathogenesis as well as experimental therapies for hereditary cerebellar ataxias. The effect of intracerebellar transplantation of embryonic cerebellar solid tissue or cell suspension on motor performance in adult Lurcher mutant and healthy wild-type mice was studied. Brain-derived neurotrophic factor level was measured in the graft and adult cerebellar tissue. Gait analysis and rotarod, horizontal wire, and wooden beam tests were carried out 2 or 6 months after the transplantation. Higher level of the brain-derived neurotrophic factor was found in the Lurcher cerebellum than in the embryonic and adult wild-type tissue. A mild improvement of gait parameters was found in graft-treated Lurcher mice. The effect was more marked in cell suspension grafts than in solid transplants and after the longer period than after the short one. Lurcher mice treated with cell suspension and examined 6 months later had a longer hind paw stride (4.11 vs. 3.73 mm, P < 0.05) and higher swing speed for both forepaws (52.46 vs. 32.79 cm/s, P < 0.01) and hind paws (63.46 vs. 43.67 cm/s, P < 0.001) than controls. On the other hand, classical motor tests were not capable of detecting clearly the change in the motor performance. No strong long-lasting negative effect of the transplantation was seen in wild-type mice, suggesting that the treatment has no harmful impact on the healthy cerebellum.

The effect of genetic background on behavioral manifestation of Grid2(Lc) mutation.

Mutant mice are commonly used models of hereditary diseases. Nevertheless, these mice have phenotypic traits of the original strain, which could interfere with the manifestation of the mutation of interest. Lurcher mice represent a model of olivocerebellar degeneration, which is caused by the Grid2(Lc) mutation. Lurchers show ataxia and various cognitive and behavioral abnormalities. The most commonly used strains of Lurcher mice are B6CBA and C3H, but there is no information about the role of genetic background on the Grid2(Lc) manifestation. The aim of this work was to compare spatial navigation in the Morris water maze, spontaneous activity in the open field and motor skills on the horizontal wire, slanted ladder and rotarod in B6CBA and C3H Lurcher mutant and wild type mice. The study showed impaired motor skills and water maze performance in both strains of Lurcher mice. Both C3H Lurcher and C3H wild type mice had poorer performances in the water maze task than their B6CBA counterparts. In the open field test, C3H mice showed higher activity and lower thigmotaxis. The study showed that genetic backgrounds can modify manifestations of the Lurcher mutation. In this case, B6CBA Lurcher mice models probably have more validity when studying the behavioral aspects of cerebellar degeneration than C3H Lurcher mice, since they do not combine abnormalities related to the Grid2(Lc) mutation with strain-specific problems.

Morphological analysis of embryonic cerebellar grafts in SCA2 mice.

SCA2 transgenic mice are thought to be a useful model of human spinocerebellar ataxia type 2. There is no effective therapy for cerebellar degenerative disorders, therefore neurotransplantation could offer hope. The aim of this work was to assess the survival and morphology of embryonic cerebellar grafts transplanted into the cerebellum of adult SCA2 mice. Four month-old homozygous SCA2 and negative control mice were treated with bilateral intracerebellar injections of an enhanced green fluorescent protein-positive embryonic cerebellar cell suspension. Graft survival and morphology were examined three months later. Graft-derived Purkinje cells and the presence of astrocytes in the graft were detected immunohistochemically. Nissl and hematoxylin-eosin techniques were used to visualize the histological structure of the graft and surrounding host tissue. Grafts survived in all experimental mice; no differences in graft structure, between SCA2 homozygous and negative mice, were found. The grafts contained numerous Purkinje cells but long distance graft-to-host axonal connections to the deep cerebellar nuclei were rarely seen. Relatively few astrocytes were found in the center of the graft. No signs of inflammation or tissue destruction were seen in the area around the grafts. Despite good graft survival and the presence of graft-derived Purkinje cells, the structure of the graft did not seem to promise any significant specific functional effects. We have shown that the graft is available for long-term experiments. Nevertheless, it would be beneficial to search for ways of enhancement of connections between the graft and host.

The role of whiskers in compensation of visual deficit in a mouse model of retinal degeneration.

Sensory deprivation in one modality can enhance the development of the remaining modalities via mechanisms of synaptic plasticity. Mice of the C3H strain suffer from RD1 retinal degeneration that leads to visual impairment at weaning age. We examined a role of whiskers in compensation of the visual deficit. In order to differentiate the contribution of the whiskers from other mechanisms that can take part in the compensation, we investigated the effect of both chronic and acute tactile deprivation. Three-month-old mice were used. We examined motor skills (rotarod, beam walking test), gait control (CatWalk system), spontaneous motor activity (open field) and CNS excitability to an acoustic stimulus for assessment of compensatory changes in auditory system (audiogenic epilepsy). In the sighted mice, the only effect was a decline in their rotarod test performance after acute whisker removal. In the blind animals, chronic tactile deprivation caused changes in their gait and impaired the performance in motor tests. Some other compensatory mechanisms were involved but the whiskers are essential for the compensation as it emerged from more marked change of gait and the worsening of the motor performance after the acute whisker removal. Both chronic and acute tactile deprivation induced anxiety-like behaviour. Only a combination of blindness and chronic tactile deprivation led to an increased sense of hearing.

Effect of dimethyl sulfoxide in cerebellar mutant Lurcher mice.

DMSO has been many times described as harmless substance, beneficial in various diseases or pathological states, including brain injury or ischemia. Using Lurcher mutant mice suffering from genetically determined olivocerebellar degeneration and normal wild type mice, we examined the effect of DMSO on spontaneous motor activity and spatial learning and orientation ability. The acute effect of DMSO was studied in mice aged 3, 6, 9 and 22 weeks. DMSO treatment decreased spontaneous activity in the open field and swimming speed in the Morris water maze in both Lurcher mutant and wild type mice. While saline-treated Lurcher mice showed age-related decline of spatial memory in the Morris water maze in DMSO-treated ones such decline did not occur. The mechanism of the effect of DMSO remains unclear. A possible explanation could be modulation of the brain perfusion and metabolism in the aging brain. The improvement of learning ability could be also mediated by a tranquilizing effect of DMSO reducing stress-induced behavioral disinhibition which is supposed to interfere with learning process in Lurcher mutants. Future studies which would investigate DMSO effects in other models of neurodegenerative diseases are necessary to verify its potential therapeutic impact.

Cerebellar degeneration in Lurcher mice under confocal laser scanning microscope.

Lurcher mutant mice represent a natural model of genetically-determined olivocerebellar degeneration caused by a mutation in the δ2 glutamate receptor gene. They suffer from progressive postnatal loss of cerebellar Purkinje cells and a decrease of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. A confocal laser scanning microscope was used aiming investigation the dynamics of changes in the cerebellar cortex of Lurcher and wild type mice derived from two strains during the period of 8-21 postnatal days. Fluorescent double-staining was used to visualize mainly the Purkinje cells in cerebellar slices. In wild types, only normal Purkinje cells of round or regular drop-shaped were present, when staining intensity of other individual cell structures differed in dependence on the age of the animal. In Lurcher mutants, there were still some normal-shaped cells. Nevertheless, depending on the animal's age, a wide variety of stages of the cell degeneration were depicted. The main characteristics of Purkinje cell degeneration in the early stage are: disruption of the continuity of the Purkinje cell layer, dark spots in cell nuclei and an irregular coloring of the cytoplasm. Later, the cells and their nuclei were deformed, often with two main dendrites sprouting from the cell body. Finally, the cell and nucleus margins were unclear, dendrites were significantly thickened, showing signs of shrinkage and fragmentation. Cell nucleoli underwent changes in number and appearance. No differences between the Lurcher mice of both strains (C3H and B6CBA) under examination were found.

Maternal infanticide and low maternal ability in cerebellar mutants Lurcher.

OBJECTIVE: One of the common, but less studied deficiencies in mouse models of cerebellar disorders is impaired breeding capacity. Nevertheless, there is no extensive study in Lurcher (Grid2Lc) mice, a model of olivocerebellar degeneration. The aim of this work was to analyze a breeding capacity of these mutants. METHODS: Lurcher females mated with healthy wild type males were compared with two control groups: wild type females mated with wild type males and wild type females mated with Lurcher males. The breeding capacity of Lurcher mice was analyzed using a fertility rate, mating capability and pups survival rate through three consecutive litters. RESULTS: Lurcher dams did not show significantly reduced fertility and mating capability. Nevertheless, their breeding capacity was affected by reduced litter size, maternal infanticide and higher pup mortality during the maternal care period. CONCLUSION: Lurcher mice are fertile and mating capable cerebellar mutants, but their breeding capacity is reduced due to the postpartum behavioral abnormalities. With regard to hyper-reactivity of the hypothalamo-pituitary-adrenal axis followed by behavioral disinhibition during stressful events in Lurcher mutants, we hypothesize that the lower breeding capacity is associated with these endocrine and behavioral abnormalities.

Intracerebellar application of P19-derived neuroprogenitor and naive stem cells to Lurcher mutant and wild type B6CBA mice.

BACKGROUND: Neurotransplantation has great potential for future treatments of various neurodegenerative disorders. Preclinically, the Lurcher mutant mouse represents an appropriate model of genetically-determined olivocerebellar degeneration. The aim of the present study was to assess survival of naïve and neurally differentiated P19 carcinoma stem cells following transplantation into the cerebellum of Lurcher mice and wild type littermates. MATERIAL/METHODS: Adult normal wild type (n=51) and Lurcher mutant mice (n=87) of the B6CBA strain were used. The mean age of the animals at the time of transplantation was 261.5 days. Suspension of naive and neurally differentiated P19 carcinoma stem cells was injected into the cerebellum of the mice. In the Lurcher mutants, 2 depths of graft injection were used. Three weeks after implantation the brains of experimental animals were examined histologically. RESULTS: Survival of neuroprogenitor grafts at a depth of 1.6 mm was significantly higher in wild type vs. Lurcher mutant mice. In wild type mice, the typical graft localization was in the middle of the cerebellum, whereas in Lurcher mice the graft was never found inside the degenerated cerebellum and was primarily localized in the mesencephalon. CONCLUSIONS: We conclude that the appearance and low survival rate of cerebellar P19 carcinoma stem cell grafts in the Lurcher mutant mice weigh against the therapeutic value of this cell line in preclinical studies of neurodegeneration.

Long-term survival of solid embryonic cerebellar grafts in Lurcher mice.

Lurcher mutant mice represent a natural model of olivocerebellar degeneration. They serve as a tool to study pathogenesis, the functional impact of the degeneration as well as therapeutic approaches. Wild type littermates are used as healthy controls. Neurotransplantation may be a promising method of therapy for neurodegenerative diseases. The aim of this work was to compare the long-term survival rate of the solid embryonic cerebellar graft in adult Lurcher mutant and wild type mice of the B6CBA strain and to assess the fundamental structural features of the graft. The graft was obtained from 12-day-old GFP mouse embryos. The brains of host mice were examined histologically 6 months after the transplantation. The graft was identified according to its GFP fluorescence. The graft presence and structure was assessed. The graft survived in all 14 Lurcher mice and in 12 of the 14 wild type mice. Cell migration and fibre sprouting from the graft were poor. No marked differences in the graft morphology between Lurcher mutant and wild type mice were found. The graft survival and appearance were similar to those after a shorter period described in a previous study. This suggests that during the 6 months, no intensive or commonly occurring processes changing the graft had proceeded and that the Lurcher mutant cerebellum niche had no strong influence over the fate of the solid cerebellar graft.

Evaluation of lipofuscin-like pigments as an index of lead-induced oxidative damage in the brain.

This study was carried out to investigate the role of lead in the development of oxidative stress in the brain. We examined the rate of lipid peroxidation and we determined lipid fluorescence products (lipofuscin-like pigments - LFP) as a marker of lipid peroxidation after short in vitro incubation of rat brain homogenates with lead acetate (10(-2), 10(-4), 10(-6) M lead acetate, 2 h). Simultaneously we examined by the same method in vivo indices of oxidative stress in brains of mice exposed for 12 weeks to 0.2% lead acetate in drinking water. The results show that the concentration of LFP in rat brain homogenates increased significantly after 2 h incubation with 10(-2) M lead acetate as compared to controls (P<0.0001). This effect was not observed in lower doses of lead acetate (10(-4) and 10(-6) M). After the long-term exposure of mice to 0.2% lead acetate, pronounced accumulation of lead and significantly increased concentration of LFP (P<0.004) in the brains of exposed animals as compared to controls were observed. The evidence for the formation of specific fluorophores originating from oxidative damage was shown also in qualitative changes in 3D spectral arrays and synchronous spectra. The presented results proved the influence of lead on the activation of radical reactions in the brain after short in vitro exposure of rat brain as well as within long-term in vivo exposure in mice using lipofuscin-like pigments as an indicator of oxidative stress.

Behavioral characteristics, associative learning capabilities, and dynamic association mapping in an animal model of cerebellar degeneration.

Young adult heterozygous Lurcher mice constitute an excellent model for studying the role of the cerebellar cortex in motor performance-including the acquisition of new motor abilities-because of the early postnatal degeneration of almost all of their Purkinje and granular cells. Wild-type and Lurcher mice were classically conditioned for eyelid responses using a delay paradigm with or without an electrolytic lesion in the interpositus nucleus. Although the late component of electrically evoked blink reflexes was smaller in amplitude and had a longer latency in Lurcher mice than that in controls, the two groups of animals presented similar acquisition curves for eyeblink conditioning. The lesion of the interpositus nucleus affected both groups of animals equally for the generation of reflex and conditioned eyelid responses. Furthermore, we recorded the multiunitary activity at the red and interpositus nuclei during the same type of associative learning. In both nuclei, the neural firing activity lagged the beginning of the conditioned response (determined by orbicularis oculi muscle response). Although red nucleus neurons and muscle activities presented a clear functional coupling (strong correlation and low asymmetry) across conditioning, the coupling between interpositus neurons and either red nucleus neurons or muscle activities was slightly significant (weak correlation and high asymmetry). Lurcher mice presented a nonlinear coupling (high asymmetry) between red nucleus neurons and muscle activities, with an evident compensatory adjustment in the correlation of firing between interpositus and red nuclei neurons (a coupling with low asymmetry), aimed probably at compensating the absence of cerebellar cortical neurons.

Ataxic gait analysis in a mouse model of the olivocerebellar degeneration.

Lurcher mutant mice represent a model of olivocerebellar degeneration. Postnatally, a complete loss of Purkinje cells and secondary reduction of granule cells and inferior olive neurons occurs. Cerebellar ataxia is among the symptoms of degeneration of the cerebellum. The aim of the work was to identify gait parameters which are changed in Lurcher mice due to cerebellar ataxia arising from functional cerebellar decortication, and to assess the correlation between gait parameters, walking speed and performance in rotarod test. We used the adult Lurcher mutant and wild type mice of the B6CBA strain. For gait analysis the CatWalk system was used. Motor functions were examined with the rotarod. Data analysis revealed significant differences between Lurchers and controls in many gait parameters. However, almost all parameters correlated with the walking speed and the differences disappeared after the correction to the walking speed. The question is what is the primary change in Lurchers-whether the walking speed or individual gait parameters. In the rotarod test, the Lurcher mutants revealed significantly worse results than the wild type animals. No correlation between the gait parameters and performance in the rotarod test was found.

A preliminary study of solid embryonic cerebellar graft survival in adult B6CBA Lurcher mutant and wild type mice.

Lurcher mutant mice represent a model of olivocerebellar degeneration. They suffer from complete loss of Purkinje cells and a reduction of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. The aim of the work was to compare solid embryonic cerebellar graft survival within a period of 9 weeks after their transplantation in adult Lurcher mutant and wild type mice of the B6CBA strain. The solid grafts were injected through a hole in the occipital bone. Host mice were sacrificed 3, 6, or 9 weeks after the transplantation and their cerebella and brain-stems were examined histologically to assess graft presence and structure. We did not find significant differences in graft survival rates between Lurcher mutant and wild type mice. The frequency of graft presence did not differ between mice examined 3, 6, and 9 weeks after the transplantation, neither in Lurchers nor in wild type mice. The grafts were of various sizes. In some cases, only small residua of the grafts were found. Nerve fiber sprouting and cell migration from the graft to the host tissue were observed more often in wild type mice than in Lurchers when examined 6 weeks after surgery. In the period 3-9 weeks after transplantation, massive dying out of the grafts was not observed despite regressive processes in some of the grafts. The degenerative changes in the Lurcher mutant cerebellum do not have strong impact on the fate of the solid cerebellar graft.

Metal, EMF, and brain energy metabolism.

Some implications of cooperative potential of metal ions and electromagnetic fields' radiation (EMF) in carcinogenic processes are discussed. It is known that these factors, chemical and physical individually have connections with processes of oxidative stress. Special attention was paid to possible manifestation within the brain. Therefore, the entry of a few potentially neurotoxic metals into the brain is discussed.

The effect of cerebellar transplantation and enforced physical activity on motor skills and spatial learning in adult Lurcher mutant mice.

Lurcher mutant mice represent a model of olivocerebellar degeneration. They are used to investigate cerebellar functions, consequences of cerebellar degeneration and methods of therapy influencing them. The aim of the work was to assess the effect of foetal cerebellar graft transplantation, repeated enforced physical activity and the combination of both these types of treatment on motor skills, spontaneous motor activity and spatial learning ability in adult B6CBA Lurcher mice. Foetal cerebellar grafts were applied into the cerebellum of Lurchers in the form of solid tissue pieces. Enforced motor activity was realised through rotarod training. Motor functions were examined using bar, ladder and rotarod tests. Spatial learning was tested in the Morris water maze. Spontaneous motor activity in the open field was observed. The presence of the graft was examined histologically. Enforced physical activity led to moderate improvement of some motor skills and to a significant amelioration of spatial learning ability in Lurchers. The transplantation of cerebellar tissue did not influence motor functions significantly but led to an improvement of spatial learning ability. Mutual advancement of the effects of both types of treatment was not observed. Spontaneous motor activity was influenced neither by physical activity nor by the transplantation. Physical activity did not influence the graft survival and development. Because nerve sprouting and cell migration from the graft to the host cerebellum was poor, the functional effects of the graft should be explained with regard to its trophic influence rather than with any involvement of the grafted cells into neural circuitries.