Immunity - a significant pathogenic factor as well as an integral part of the psychoneuroendocrine-immune regulations.

Immunity plays an important role in the reactivity of the organism and, in this context, is an essential factor in the pathogenesis of many diseases. Basically, there is no system or organ in the body, whose dysfunction is not related to immunity consequences. In addition, there are also multisystem diseases simultaneously involving multiple body systems. They are not always caused by weak immunity, but also often by modified immune reactions known as overshooting. The essence of all these diseases is a change in the reactivity of the organism where immunity plays an important role. The immunity as such is then part of the systems of neuroendocrine-immune regulation, which have common mediators and receptors. The establishment of psychoneuroimmunology, a relatively new discipline in neuroscience, contributed to a detailed understanding of these mechanisms between central and peripheral nervous system, the endocrine system and the immune system. This research enabled the uncovering of the nature of stress-diseases and impact of other regulatory disturbances on the function of various body organs and systems of the organism as a whole. The aim of this short review is to show complex interconnections of these relationships to better understand the human health and disease.

Twenty years of cerebellar degeneration research at the department of pathological physiology, faculty of medicine, Pilsen, Charles University.

Mutant Lurcher mice represent an animal model of naturally occurring cerebellar degeneration. A gene mutation causes the demise of all Purkinje cells, as along with certain other types, as well as the functional elimination of the cerebellar cortex. Involvement in the research using this model of the C3H strain began at the Department of Physiology, UCL in 1995/96. It continued in scientific cooperation with other European laboratories where we obtained Lurcher mice of the B6CBA strain. The aim of the effort was first to identify the extent to which the cerebellum is involved in the higher nervous activity, i.e. cognitive and other functions. In that research, use was made of an entire array of methodological procedures to examine learning, memory, motor functions and emotional behavior. It was completed with an electrophysiological examination of the brain and special microscopic procedures. The results demonstrated that the cerebellum (aside from its traditional tasks) does in fact play a significant role in cognitive function, emotions, etc. It was further found that the neurodegenerative processes also affected the immune and endocrine functions, confirming the concept of the unity of the psycho-neuroendocrine-immune system. Surprisingly, despite their neurological impairment, the affected animals were able to learn to some extent and, make progress with physical training, improving not only their motor skills but also learning and memory, including deferring of signs of aging. These particular findings may prove useful for human medicine.

Jan Evangelista Purkyne and the cerebellum then and now.

The name of Jan Evangelista Purkyne and the cerebellum belong inseparably together. He was the first who saw and described the largest nerve cells in the brain, de facto in the cerebellum. The most distinguished researchers of the nervous system then showed him the highest recognition by naming these neurons as Purkinje cells. Through experiments by J. E. Purkyne and his followers properly functionally was attributed to the cerebellum share in precision of motor skills. Despite ongoing and fruitful research, after a relatively long time, especially in the last two decades, scientists had to constantly replenish and re-evaluate the traditional conception of the cerebellum and formulate a new one. It started in the early 1990s, when it was found that cerebellar cortex contains more neurons than the cerebral cortex. Shortly thereafter it was gradually revealed that such enormous numbers of neural cells are not without an impact on brain functions and that the cerebellum, except its traditional role in the motor skills, 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 below.

Comparison of P19-derived neuroprogenitor and naive cell survival after intracerebellar application into B6CBA mice.

Mouse embryonic carcinoma cells (P19 line) were studied for both their survival and developmental potential in the intact cerebellum of B6CBA mice. The P19 cells were cultured and labelled with green fluorescent protein using transfection. Cells were used for transplantation either in the undifferentiated stage or after 3 days of neurodifferentiation induced by retinoic acid. The intracerebellar application was performed in 43 mice: group A (N = 21) received neuroprogenitors and group B (N = 22) received undifferentiated cells. The morphology of transplanted cells within the context of the surrounding cerebellar tissue was evaluated after 3 weeks. Naive P19 cells engrafted and survived in the cerebellum of 7 of the 22 adult mice (survival rate 31.8 %). Neuroprogenitors survived in 13 of the 21 mice (survival rate was 61.9 %). Since the cut-off is P < 0.05, the difference is not statistically significant (P = 0.069). An expansive appearance of the graft was significantly more frequent (P = 0.0047) in naive P19 cells than in neuroprogenitors. In mice in which the grafts did not survive, no marks of grafted cells or only fluorescing detritus were found. In conclusion, this is the first study to track the fate and morphology of embryonic carcinoma cells transplanted into the cerebellum, confirming that neuroprogenitors derived from embryonic carcinoma cells can settle in the host tissue and differentiate according to the surrounding conditions. With further validation, the embryonic carcinoma cells could become a valuable model with which to study the impact of cell therapy on neurodegenerative diseases.

Characterization of P19 cells during retinoic acid induced differentiation.

The aim of our study was to characterize mouse embryonal carcinoma (EC) cells P19 in different stages of retinoic acid induced neurodifferentiation by two methods, immunocytochemistry and RT qPCR. The characterization of the cells is crucial before any transplantation into any model, e.g. in our case into the mouse brain with the aim to treat a neurodegenerative disease. Specific protein markers (MAP-2, OCT-4, FORSE-1) were detected by immunocytochemistry in the cell cultures. The mRNA expression levels of PAX-6, MASH-1, Brachyury, GATA-4 and AFP were determined by RT qPCR method. HPRT was used as a housekeeping gene. The degree of differentiation can be characterized by expression of analyzed genes. The presence of OCT-4 and FORSE-1 proteins in undifferentiated pluripotent cells and the presence of dendrite specific MAP-2 in neuroprogenitors was detected. The expression levels of PAX-6 and MASH-1 increased and expression of Brachyury decreased during the neurodifferentiation process. The expression levels of GATA-4 and AFP were the highest after induction of differentiation with retinoic acid. Detailed characterization of cells before transplantation experiments can contribute to better understanding of their effect.

Glutamate receptor block in Lurcher mutant mice during ontogeny and its effect on hippocampal long-term potentiation.

Basic evaluation of the effect of chronic NMDA glutamate receptor (NMDAR) blockade on the hippocampal long-term potentiation (LTP) was performed in an animal model of inborn olivo-cerebellar degeneration (Lurcher mutant mice, LMM). NMDA receptor antagonist MK-801 was administered to mice in the dose 0.2 mg/kg of body weight, daily during two periods of their ontogeny: D5-D26 and D91-D111. In the consecutive 15 days some behavioral characteristics were studied using special methods for physical activity testing. Then LTP was investigated in LMM and also in their healthy littermates which served as controls (wild-type, WT). LTP in animals pre-treated with MK-801 showed significant long-term suppression of NMDAR activity, in both WT and LMM despite certain small differences between them. Our results show that cerebellar pathology on one hand and a physical activity on the other hand can influence the LTP in hippocampal region. It can be concluded that the results support the ideas of close functional cooperation between the brain structures which are involved in mechanisms of learning and memory.

Changes of motor abilities during ontogenetic development in Lurcher mutant mice.

Lurcher mutant mice represent a natural model of olivocerebellar degeneration. This degeneration is caused by a mutation of the gene for the delta2 glutamate receptor. Lurcher mutants suffer from cerebellar ataxia and cognitive functions deficiency as a consequence of excitotoxic apoptosis of Purkinje cells in the cerebellar cortex and a secondary decrease of granule cells and inferior olive neurons. This process finishes by the 90th day of postnatal life, but already by 14 days, the Purkinje cells are damaged and the ataxia is fully developed. Purkinje cells die by apoptosis within the first 3 weeks of life. The aim of our work was to study the development of motor functions in the course of the ontogenetic development in Lurcher mutant mice of the B6CBA strain and to compare it with wild type mice of the same strain. Mice aged 2, 3, 6, 9, and 22 weeks were used in our experiment. Motor skills were examined using four standard tests: the horizontal wire, rotating cylinder, footbridge and slanting ladder. Our findings in Lurcher mutant mice show a significant increase of motor abilities up to the sixth postnatal week and selective decrease early after this period. This improvement of motor skills is caused by the physiological development of musculature and the nervous system, probably with some contribution of plasticity of the maturing brain. The cause of the decline of these abilities immediately after the completion of the development is unknown.

Hippocampal long-term potentiation in adult Lurcher mutant mice: effect of embryonic cerebellar graft and motor training.

Possible effect of trophic factors from embryonic cerebellar graft transplanted in adult Lurcher mutant mice on LTP as electrophysiological marker of learning and memory process was studied. Also the combination of the transplantation and long-term forced motor training was investigated. An evaluation of LTP ability in four animal groups (transplanted, sham-operated, with and without forced motor activity) and comparison among them showed the highest LTP improvement in the group with combination of both influences (ie. transplantation and motor training).

The effect of D1-like receptor blockade on motor functions and spatial learning in B6CBA mice.

Dopaminergic neurotransmitter system plays a role in many neural functions, for instance in the motor system. Dopamine also influences cognitive functions, motivation and behaviour. In this study, the effect of D1-like receptors blocker SCH 23390 on spontaneous motor activity, motor functions and spatial learning in adult wild type mice derived from the strain B6CBA was investigated. The animals of one group were administered with D1-like receptors blocker SCH 23390 while the mice from the other group were treated with the saline solution 20 minutes before experiments. Spontaneous motor activity was examined in the open field. Motor functions were examined using horizontal wire, ladder and rotarod. Spatial learning was tested in the Morris water maze. D1-like receptors inhibition significantly decreased spontaneous motor activity. In the horizontal wire test, the mice treated with SCH 23390 manifested significantly better results than control animals. No significant differences between experimental animals and controls were found in the ladder test. On the rotarod, the animals with D1-like receptors inhibition reached significantly shorter latencies than the animals treated with the saline solution. D1-like receptors blockade led to significant impairment of performance in experimental animals in the Morris water maze. Spontaneous motor activity and motor skills were influenced by evoked hypokinesia. Meanwhile in the horizontal wire test this effect seemed to be advantageous, in the rotarod test it meant a definite handicap because this test requires good motor coordination and activity. The results gained in the Morris water maze indicated that not only hypokinesia played some role there but also spatial learning and perhaps motivation were affected. The study confirmed that D1-like receptors inhibition has effects on both motor and cognitive functions in mice. However the motor effects are not the main cause of the failing in the spatial learning test.

The effect of nitric oxide synthase inhibitors nitro-L-arginine and 7-nitroindazole on spatial learning and motor functions in Lurcher mutant and wild type mice.

Nitric oxide (NO) is an intercellular messenger that, among other things, plays an important role in the nervous system as a gaseous neurotransmitter, modulating long-term potentiation (LTP) induction of synaptic transmission. LTP has been suggested to be the basis of memory formation. On the other hand NO also participates in excitotoxic processes which play an important role in many neuropathological states. The aim of this work was to observe the effect of two NO synthase (NOS) inhibitors (N omega-Nitro-L-arginine, NA; 7-nitroindazole, NI) on spontaneous behaviour, spatial learning and motor functions in Lurcher (+/Lc) and wild type (+/+) mice, derived from the B6CBA strain. Heterozygous Lurcher mutant mice represent a natural model of the olivocerebellar degeneration. They suffer from postnatal, practically total, extinction of cerebellar Purkinje cells (due to the excitotoxic apoptosis) and a partial decrease of granule cells and inferior olive neurons (ION) because of the lost target of their axons. +/+ animals are healthy littermates of +/Lc. NA is a nonselective NOS inhibitor which influences, except neuronal (n), also endothelial (e) NOS with an impact on blood pressure, NI is a selective nNOS inhibitor without any circulatory effect. The adult animals of both types (+/Lc; +/+) were influenced by acute administration of both inhibitors (25 mg/kg i.p. 30 min. before experiments) and newborns only by both acute and long-term administration of NI (1 month, starting from postnatal day 2, P2). Control solutions - saline or solvents of both NA and NI inhibitors--diluted 1M HCl and dimethyl sulfoxide (DMSO) respectively, were given at a relevant volume in the same way. The effect of both inhibitors and control solutions on motor functions was tested using four standard procedures (horizontal wire, slanting ladder, rotating cylinder, foot-bridge); in newborns at the age of 14 days. Spatial learning ability was examined in five-day long procedure in the Morris water maze (MWM) (in newborns started on P21). Spontaneous behaviour was studied only in adult animals (after acutely influencing them) employing the open field method. The results showed, that neither the Lurcher mutant, nor wild type mice derived from the B6CBA strain were significantly affected by NOS inhibitors NA and NI in spatial learning after both the acute and long-term application. Only significant decrease of swimming speed was found in both types of mice after the acute administration of NI and in the wild type animals after the acute administration of NA. Motor functions were significantly negatively affected only in the Lurcher mutants after both the acute and chronic application of NI.

The time-dependent block of NMDA glutamate receptor influences hippocampal LTP in inborn cerebellar degeneration mouse model.

The effect of single dose of NMDA glutamate receptor blockage administration on the hippocampal LTP was evaluated in animal model of inborn cerebellar degeneration. We compared the level of possible LTP blockade in two groups of animals, Lurcher mutant mice and their healthy littermates which served as controls. In the second part of the study we tested group of mice which were influenced repeatedly by the same NMDA blocker (MK-801) during behavioral experiments. Our results suggest a similar effect of blockade either after single or chronic MK-801 administration; both of them practically disrupted LTP generation with differences between healthy and neurodegenerative animals.

Preliminary study of the effect of repeated motor training on spatial learning ability in adult lurcher mutant mice.

Lurcher mutant mice represent a model of olivocerebellar degeneration. They suffer from cerebellar ataxia and deterioration of cognitive functions. The aim of the work was to study the effect of repetitive enforced motor training on spatial learning ability and motor coordination in adult Lurcher mutant mice of the C57BI strain. Experimental mice were trained repetitively on a rotarod. Control mice were left without the training. Motor coordination was tested four times-before the training, in the third week of the training, at the end of the training and after a spatial learning test following the training. A rotarod of higher cylinder diameter and lower rotation speed was used. Spatial learning was examined using the Morris water maze. Trained animals achieved significantly better results than untrained mice in the 2nd and 3rd motor coordination test. In the last test following the spatial learning examination, untrained mice improved their performances so that there were no differences between trained and untrained group. In the Morris water maze trained mice showed higher spatial learning ability than untrained animals. Motor coordination capability of adult Lurcher mutant mice was improved by the training on rotarod but also by swimming during the experiment in the water maze. Repetitive motor activity led to increase of spatial learning ability.

Changes of dopamine receptors in mice with olivocerebellar degeneration.

Lurcher mutants are mice with functional mutation in the 82 glutamate receptor (GluRdelta2) that is predominantly expressed in cerebellar Purkinje cells and plays a crucial role in cerebellar functions. These mice display ataxia and impaired motor-related learning tasks. In order to elucidate the role of dopaminergic receptor system in coping with mutation in delta2 glutamate receptor the behavioral effect (spatial learning) of D1 dopamine receptor activation and inhibition and changes in D1-like and D2-like dopamine receptors in striatum, hippocampus and cerebellum in C57BI/7 and C3H Lurcher mutants and wild type mice were studied. We have found that Lurcher mutants were worse in the spatial learning but mice of both types reacted similarly to D1 dopamine receptor agonist (without effect) and antagonist (worsening). Moreover, Lurchers revealed substantial higher density of both D1-like and D2-like dopamine receptors in hippocampus in C57BI/7 strain, while in C3H strain only D1-like dopamine receptors were higher. In C57BI/7 strain, D-like dopamine receptors were lower in cerebellum; D2-like dopamine receptors were not affected. In the striatum, the receptor densities were similar to the wild type counterparts. Our results suggest specific participation of dopamine receptor system in coping with olivocerebellar degeneration.

Does transplantation of cerebellar embryonic tissue influence hippocampal LTP in adult Lurcher mutant mice?

Possible influence of embryonic cerebellar graft transplanted into the adult neurodegenerative brain in Lurcher mutant mice on long-term potentiation (LTP) in hippocampus was investigated. Evaluation of LTP ability and comparison with the tests of motor learning suggests similarities between magnitude of LTP and criteria of motor learning. Also interstrain differences were described. Our results support ideas about tight cooperation among brain structures which are involved in mechanisms of learning and memory.

Comparison of embryonic cerebellar graft survival in adult Lurcher mutant mice of strains C3H and C57Bl/7.

Lurcher mutant mice suffer from complete loss of cerebellar Purkinje cells. The aim of the work was to compare the solid embryonic cerebellar graft survival in adult Lurcher mutant mice derived from strains C3H and C57Bl/7 and to assess the morphology of the grafts. Embryonic cerebellar tissue was obtained from 12-13 days mice embryos expressing green fluorescent protein (GFP). Embryonic cerebellum was injected with a glass microcapillary into the cerebellum of adult Lurcher mutant mouse. Host mice were sacrificed 2-12 weeks after the transplantation. Brainstems and cerebella were examined histologically. The graft and graft derived GFP-positive cells were detected according to their green fluorescence. To visualise the structure of the graft Nissl staining was used. Graft survival percentage was evaluated in groups of mice sacrificed during the first, second or third month after the transplantation. The graft was found in all C57Bl/7 mice and in 90.9% of C3H mice examined within one month after the transplantation. In the second month the graft was present in 83.3% of C57Bl/7 and 50.0% of C3H mice. Till the third month the graft survived in 68.2% of C57Bl/7 mice and 22.2% of C3H mice. In C57Bl/7 mice a cerebellar structure was developed in the graft and migration of graft derived-cells to the host tissue was observed more often than in C3H mice. C567Bl/7 mice seem to be more suitable for experiments testing functional consequences of transplantation into the cerebellum requiring good long-term graft survival.

Neural functional and morphological consequences of retinal degeneration in C3H Lurcher mutant and wild type mice.

Lurcher mutant mice represent a model of olivocerebellar degeneration associated with the total functional elimination of the cerebellar cortex. The affected animals suffer from cerebellar ataxia and worsening of cognitive functions. Healthy littermates of Lurchers-wild type mice serve as controls. Except mentioned patterns some animals derived from the C3H strain exhibit signs of a hereditary retinal degeneration. The impact of the retinal degeneration on visuospatial abilities and on the neuronal morphology in visual projection of both C3H Lurcher mutant and wild type mice has been studied in this work. The Morrris water maze was used for examination of spatial learning when the animals learned to find a platform hidden under the water surface. Time of reaching the platform (escape latency) in individual experimental days as well as the swimming velocity was measured and the strategy of maze exploration was assessed. The presence of the retinal degeneration was proved histologically by means of classical hematoxillin-eosin method. The neurohistological examination of the superior colliculus and visual cortex was performed using a Ramón-Moliner modification of the Golgi method. The results obtained showed that retinal degeneration influenced the strategy of the maze exploration and caused generally worse results. The histological examination of eyes in animals with bad results confirmed presence of the retinal degeneration. The neurohistological examination of the brain visual projections of animals affected with the retinal defect showed most detectable changes in dendritic spines of the V1 cortex (lower density in general and less immature types).

Various methods of Purkinje cells transplantation and their functional response in Lurcher mutant mice.

Embryonic cerebellum was transplanted to adult Lurcher mutant mice affected with hereditary olivocerebellar degeneration and with resulting cerebellar ataxia. Grafts were applied as solid pieces of tissue or as cell suspensions. The aim was to replace Purkinje cells lost by the neurodegeneration with embryonic cells and to observe the effect on motor symptoms of cerebellar ataxia. Success rate of the two methods was also compared. Motor skills were tested before and in week intervals after the transplantation. The results were compared with sham-operated controls. When the solid graft was transplanted, the success rate was two times higher as compared with the cell suspension method. Fibre sprouting and cell migration from the graft to the host tissue was observed. Insignificant amelioration of motor skills was found in mice after the solid cerebellar tissue transplantation, while the cell suspension application had no effect.

Functional consequences of retinal degeneration in spatial orientation in C3H wild type and Lurcher mutant mice.

Lurcher mutant mice represent a model of genetically determined olivocerebellar degeneration. In the C3H strain there is also hereditary retinal degeneration. The aim of this work was to assess, whether the retinal degeneration influences spatial orientation and results of the spatial learning tasks. Two experiments in the Morris water maze were arranged. First, mice learned to find a platform position, which was linked to two labels on the periphery of the maze. In the second experiment the platform was removed and swimming velocity and preference of central or peripheral zone of the maze were assessed. Presence of the retinal degeneration was detected histologically. Both Lurcher mutant and wild type mice that exhibited long latencies in the first experiment were affected with the retinal degeneration, while animals that performed the trial well, had normal retina. Swimming velocity was not changed substantially. The maze exploration strategy was different in mice with and without the retinal degeneration.

Effect of whole-body exposure to high-frequency electromagnetic field on the brain electrogeny in neurodefective and healthy mice.

A direct registration of brain cortical and hippocampal activity during a high-frequency electromagnetic field (HF EMF) exposure was performed. All experimental procedures were done under urethane anaesthesia (20%, 2 g/kg i.p.) in Lurcher mutant mice, wild type (healthy littermates) were used as controls. Experimental animals were exposed to the HF EMF with frequency corresponding to cellular phones. Our method is based on the use of gel electrodes (silicon tubes or glass microcapillaries filled with agar) where the connection with classical electrodes is located out of HF EMF space. ECoG evaluation showed a distinct shift to lower frequency components but clear effect has been observed only in wild type (healthy) mice whereas in Lurcher mutant mice only gentle differences between frequency spectra were found. Measurement of hippocampal rhythmicity showed gentle changes with increase of higher frequencies (i.e. opposite effect than in cortex) and changes in theta oscillations registered from a dentate gyrus and CA1 area in both types of animals (healthy and mutant). These findings support the idea about possible influencing the central nervous system by HF EMF exposure and support also some recent results about possible health risks resulting from cellular phones use.