Neuroglia - development and role in physiological and pathophysiological processes.

The dynamic development of studies on neuroglia in recent years indicates its previously underestimated role in maintaining proper brain function, both in physiological and pathological conditions. The use of modern research methods such as single-cell techniques as well as in vivo and in vitro models enriched the state of our knowledge. The most important issues regarding the maturation and development of neuroglia include cooperation between glial cell groups and with neurons in neurogenesis, neuroregeneration, (re)myelination and how the early developmental roles of glia contribute to nervous system dysfunction in neurodevelopmental and neurodegenerative disorders. There is still growing evidence emphasizing the importance of astroglia in maintaining the brain physiological homeostasis, regulation of immune response, cerebral blood flow, and involvement in the reactive neurogliosis, precisely adapted to the nature of pathological stimulus and the depth of tissue damage. The important issues related to the function of oligodendrocytes include explanation of the mechanisms of interaction between the glial cells and myelinated axons, important not only in myelination, but also in development of cognitive processes and memory. Further studies are required for understanding the mechanisms of demyelination occurring in several central nervous system (CNS) diseases. An interesting area of research is related with explanation of the NG2 glia function, characterised by significant proliferative potential and ability to differentiate in both in physiological conditions and in pathology, as well as the presence of synaptic neural-glial connections, which are especially numerous during development. The increasing knowledge of microglia comprises the presence of specialised subsets of microglia, their role the myelination process and neurovascular unit functioning. We are only beginning to understand how microglia enter the brain and develop distinct functional states during ontogeny. This review summarises the current state of knowledge on the development and role in the CNS of different, heterogeneous cell populations defined by a common term neuroglia.

Can the kisspeptin help us in the understanding of pathology of some neurodegenerative brain diseases?

It is already known that the discovery of kisspeptin was a revolutionary step in the understanding of neuroendocrine regulation of reproduction. Kisspeptin is one of the main moderators of the gonadotropic axis, but the kisspeptin gene is known to be expressed in various regions of the central nervous system. The activity of kisspeptin is not limited to hypothalamic pituitary gonadal axis; it participates in the regulation of multiple neuronal circuits in the limbic system. The limbic system is a part of the brain involved in behavioural and emotional reactions, and disturbances in its functioning may be the source of some psychiatric as well as degenerative disorders. In the present review, we summarise the current state of knowledge concerning the role of kisspeptin in the limbic system and a new hope for the treatment of disturbances in its functioning.

Role of brain-derived neurotrophic factor in shaping the behavioural response to environmental stressors.

Brain-derived neurotrophic factor (BDNF) is an important neurotrophin involved in an integration of the brain activity in physiological and pathological conditions, with formation of a short- and long-term functional and structural neuroplasticity. This process proceeds, with a changeable dynamics, in the subsequent stages of ontogenesis. In addition to many other functions in the central nervous system, BDNF is also involved in shaping a response to stress stimuli in the form of precisely adjusted behavioural reactions involving the limbic system, and the endocrine system with stimulation of the hypothalamic-pituitary-adrenal axis (HPA). Although almost every stressor increases the activity of the HPA, the neuronal response to it can vary substantially. This may be due to involvement of different neurotransmitter pathways, neuromodulators and neurohormones, as well as changes in gene expression. It is widely accepted that BDNF synthesis and secretion are modulated by stress. Furthermore, age is an important factor influencing the BDNF expression in response to different stressors. In this work, we focused on the analysis of the role of mild stressful stimuli, which commonly occur in the natural environment, on changes in BDNF expression at various stages of ontogenetic development. Although, the presented data comes from animal studies, probably similar mechanisms of stress regulation are also present in humans. This comprehensive review shows that the influence of stressors on the BDNF expression depends on many factors, including a type and duration of a stressor, time of neurotrophin detection, animal's resistance to stress, brain area, and genotypic characteristics of an individual. A more detailed understanding of the mechanisms shaping stress reactions, including the role of BDNF, may be of both theoretical and practical importance, allowing designing more effective strategies for preventing and treating stress itself and the stress-related disorders.

The pathophysiology of intracerebral haemorrhage.

Spontaneous intracerebral haemorrhage carries a high mortality rate and treatment of the disease raises more questions then answers. Mass effect, ischaemia and toxicity of blood components are responsible for brain tissue damage. Initially occurring disturbances of cerebral blood flow have a temporary character and do not play a key role in the pathology of intracerebral haematoma. Oedema formatting in the 24-48 hours after intracerebral bleeding is the result of multidirectional processes. The pathological mechanism that underlines it is the function of activation of systemic complement and cascade of coagulation. In the light of these findings, further clinical and experimental investigations should be focused on these factors.

The technique of inhalation anaethesia in experimental investigation in the rat.

The rat is the most frequently used animal in scientific inquiry conducted for the purpose of advancing basic knowledge that may lead to an improvement in the results of treatment. Understanding of the pharmacological properties of inhalation anaesthetics, in combination with monitoring of their concentration in the inspired and end-tidal gas, together provide safe and precise control of the depth of the anaesthesia. However, accurate application of the inhalation method of anaesthesia requires special equipment for the delivery and effective scavenging of inhalation anaesthetics.

Quantitative analysis of influence of sevoflurane on the reactivity of microglial cells in the course of the experimental model of intracerebral haemorrhage.

BACKGROUND: Microglial cells play an important role in the pathophysiology of intracerebral haemorrhage. We have examined the possible influence of sevoflurane on the reactivity of microglial cells during intracranial haemorrhage. METHODS: Forty adult male rats were divided into two groups. All animals were anaesthetized with fentanyl, dehydrobenzperidol and midazolam. In the experimental group animals additionally received sevoflurane 2.2 vol% end-tidal concentration. Intracranial haemorrhage was produced through infusion of blood into the striatum. The microglial cell population (numerical density of immunoreactive cells and their distribution) was assessed on days 1, 3, 7, 14 and 21 after producing a haematoma using antibodies OX42 and OX6. RESULTS: In the control group significant differences in the density of OX42-ir cells between 3rd and 7th (81.86 vs. 129.99) (95% CI: -77.99 to -18.25, P = 0.0035) and between 14th and 21st (105.36 vs. 63.81) (95% CI: 13.21 to 69.89, P = 0.006) survival days were observed. However, significant increase of percentage of amoeboid OX42-ir cells between 3rd and 7th (0.98 vs. 48.71) (95% CI: -52.17 to -43.30, P = 0.0001) and between 7th and 14th (48.71 vs. 58.47) (95% CI: -13.96 to -5.55, P = 0.0002) and then their decrease - between 14th and 21st (58.47 vs. 31.74) (95% CI: 22.52 to 30.93, P = 0.0001) days of observation were noted. In the sevoflurane groups OX42-ir cells were not found. On the 3rd day the density of OX6-ir cells in the sevoflurane group was significantly lower than that in the control group (12.39 vs. 34.57) (95% CI: -49.78 to -2.96, P = 0.02). The percentage of an amoeboid form of OX6-ir cells was significantly lower in the sevoflurane group than that in the control group (27.31 vs. 82.03) (95% CI: -72.52 to -36.92, P = 0.0001) (58.76 vs. 82.37) (95% CI: -38.81 to -8.41, P = 0.003) (42.87 vs. 81.55) (95% CI: -53.23 to -24.10, P = 0.0001) respectively for 3rd, 7th and 14th days of survival. CONCLUSION: Administration of sevoflurane during anaesthesia in animals with intracerebral haemorrhage evoked a decrease of activation of the microglial cells.

The influence of sevoflurane on the reactivity of astrocytes in the course of the experimental intracerebral haemorrhage in rat.

40 adult Wistar rats were divided into two groups depending on the applied anaesthesia. In both groups animals were generally anaesthetized with fentanyl, dehydrobenzperidol administered intraperitoneally and midazolam given intramuscularly. In the second group (SEVO) animals received sevoflurane of 2.2 vol% end-tidal concentration. Intracerebral haematoma was produced through infusion of 100 microl of autologous blood into the striatum. Each group was divided into five subgroups depending on the length of survival period: 1, 3, 7, 14, 21 days. The astrocytic population was studied by means of anti-GFAP staining. Stereological analysis was applied to estimate the numerical density of immunoreactive cells and the distribution of their types. On 7th day of observation the density of GFAP-immunoreactive astrocytes in SEVO was lower (p<0,05) than that in the control group. In the control group, the increase (p<0.05) of per cent of activated astrocytes between the 1st and 3rd survival day was noted, which remained at this level till the end of observation. In SEVO group, the increase (p<0.05) of per cent of activated astrocytes between the 3rd and 7th day and the decrease (p<0.05) between the 14th and 21st survival day were observed. During days of observation the per cent of activated astrocytes was lower (p<0.05) in the SEVO group than that in the control group. Administration of sevoflurane during anaesthesia to animals with intracerebral haemorrhage has evoked not only the delay of the activation of astrocytes but also decrease in its level.

Apoptosis in the course of experimetal intracerebral haemorrhage in the rat.

Intracerebral haematoma was produced in 25 adult rats by infusion of 100 microl of autologous blood into the striatum. The animals' brains were removed at 1, 3, 7, 14 and 21 days after production of the haematoma. The TUNEL method was used to detect DNA fragmentation and TUNEL-positive cells were qualified. TUNEL-positive cells were already found on the first day of observation and were present for three weeks after haematoma production. These results provide evidence that programmed cell death is associated with intracerebral haemorrhage.

Relation between the age of specimen and the shrinkage of brain frozen sections.

The aim of the study was to assess the effect of age of the animal upon the real thickness of the frozen sections. The study was performed on 19 rabbit brains. The thickness of the frozen sections regardless of their staining is age-dependent. The relation is proportional during the period from 7 to 180 postnatal day and characterizes both immunohistochemical as well as cresyl violet-stained sections; moreover, changes of the section thickness proceed parallelly. It is suggested that especially for some stereological parameters all required procedures should be standardized to achieve comparable and unbiasedly interpretable results.

Differentiation in the immunocytochemical features of intrinsic and cortically projecting neurons in the rat claustrum -- combined immunocytochemical and axonal transport study.

Retrograde axonal transport method of the fluorescent tracer FluoroGold (FG) was combined with immunocytochemistry to investigate the occurrence of nitric oxide synthase (NOS), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in both intrinsic and cortically projecting neurons of the rat claustrum. Only NOS was detected in both the scattered projecting neurons and internal neurons of the claustrum. Approximately 20% of NOS-immunoreactive neurons in the claustrum were also retrogradely labeled with FG after tracer injections into the frontal cortex. The other substances were exclusively confined to the population of interneurons, which mainly displayed an oval, round or fusiform shape and a medium size. Apart from the neuronal somata, the proximal parts of the dendritic arborization were clearly visible. The immunoreactive neurons were randomly distributed in the claustrum and their neuronal size and shape did not differ in the various parts of the studied structure. Co-localization of NOS and SOM or NOS and NPY was reported. In conclusion, SOM, VIP and NPY do not appear to play a significant role in the claustro-cortical projection but are most probably involved in modulation and information transfer in the claustrum. The appearance of NOS in both cortically projecting and intrinsic neurons of the claustrum may be indicative of a fundamentally different role in the functioning of the claustro-cortical loop.

Effect of sevoflurane on intracranial pressure and cardiovascular function in rabbits with experimental intracerebral haematoma.

BACKGROUND: Sevoflurane is one of the newest volatile anaesthetic agents. The effect of sevoflurane on ICP in conditions of intracranial pathology is essential from the clinical point of view but still not sufficiently clear. The aim of the study was to examine the effects of 1 MAC, 2 MAC, 3 MAC sevoflurane on intracranial pressure (ICP), mean arterial blood pressure (MABP) and heart rate (HR) in rabbits with experimental intracerebral haematoma (ICH). MATERIAL AND METHODS: The experiments were performed in 13 adult rabbits, 3.5-4.0 kg weight. The rabbits were randomly allocated to two different groups. In group I, (n = 7), sevoflurane was administered in stepwise increasing concentrations of 2.2 vol%, 4.4 vol% and 6.6 vol%, each for a period of 15 minutes. In group II (n = 6), intracerebral haematoma was produced and subsequently sevoflurane was administered in the same manner as in group I. Ventilation parameters, inspiratory and end-tidal sevoflurane concentration, end-tidal CO2 concentration (ETCO2), HR, MABP, ICP and body temperature, measured in the nasopharynx, were monitored throughout the experiment. RESULTS: Mean values of ETCO2 and temperature in the nasopharynx were not significantly different in both groups and remained stable in the whole observation period. In group II in all cases the evidence of intraventricular haematoma was observed. In this group mean values of ICP, MABP and HR after haematoma production were significantly higher than those in group I. Statistically significant increase of ICP was observed in 30th minute in group I, while in 35th minute in group II. In both groups a statistically significant decrease in MABP was observed from 20th minute of observation. A significant decrease in HR in both groups from 25th minute of experiment was also observed. CONCLUSION: In conclusion it should be stressed that sevoflurane, in doses not exceeding 1 MAC, shows no significant effect on ICP and cardiovascular function in the course of intracranial haematoma.

General anaesthesia in rats undergoing experiments on the central nervous system.

The rat is one of the species most commonly used in laboratory practice. Numerous publications concerning various aspects of morphology and physiology are based on the results obtained in this species. It make these results comparable and under some precautions enables to transpose into the relationships observed in humans. Each experimental project must obtain the permission of the Local Ethical Committee, as well as comply with the regulations of the European Communities Council, outlined in the "European Convention for the protection of vertebrate animals used for experimental and other scientific purposes". Adequate pre-operative care can eliminate or reduce the incidence of many complications, which may occur during anaesthesia. General anaesthesia in experimental practice can be achieved using a variety of drugs and ways of administration, among others inhalational or intravenous. The side effects of anaesthetic agents can be reduced in this way. Knowledge of the effect of anaesthetics on the cerebral circulation, metabolism and intracranial pressure in both normal and pathological conditions is crucial for neurobiological purposes. Many anaesthetic agents depress respiration, which can result in hypoxia, hypercapnia and acidosis. To maintain blood carbon dioxide and oxygen concentration in the physiological range, it is necessary to apply tracheal intubation and artificial ventilation. However, even when using sophisticated equipment, the role of basic clinical observation, such as the colour of the blood shed in the operation field, breathing depth and frequency, cannot be overestimated. The importance of monitoring mean arterial blood pressure and intracranial pressure in experiments on the central nervous system is fundamental. Special attention should be paid to controlling the temperature and monitoring the fluid balance. Appropriate postoperative care can have a decisive influence on the final results of the research.

The influence of the intraventricular hemorrhage on the intracranial pressure and hemodynamic changes in the experimental intracerebral hemorrhage in the rabbits.

The experimental model of the intracerebral hematoma in the rabbit was used for the investigation of the changes of the intracranial pressure and selected hemodynamic parameters. The study was performed on 13 adult rabbits, divided into two groups receiving 1 ml (group I--6 animals) and 2 ml (group II--7 animals) of fresh arterial blood, respectively. The monitoring of the intracranial pressure (ICP), the mean arterial blood pressure (MABP), the heart rate (HR), the end-tidal CO2 concentration (ETCO2) and the body temperature was measured every minute in the hematoma production phase and every 5 minutes for the consecutive three hours. The volume of the hematoma was calculated according to Cavalieri formula, with the use of the system for the automatic picture analysis. The mean volume of the intraparenchymal part of the hematoma in group I was higher than in the group II. However, in all the representatives of the second group the evidence of the intraventricular hemorrhage was present. The dynamics of the ICP, MABP and HR changes differed significantly in both groups during the period of the observation. On the basis of the physiological and morphological observations we conclude that the changes of ICP remain the most sensitive and valuable parameter during the early course of the intracerebral hemorrhage. Coexistence of the rapid ICP, MABP and HR changes must be always regarded as the possible sign of the intraventricular hemorrhage.

The different patterns of maturation of the claustrocortical connections in a rabbit.

The quantitative analysis of the claustrocortical connections labeled with the fluorescent retrograde tracer Fluoro-Gold (FG) was conducted on 90 rabbits subdivided into the following age groups (P2, P7, P14, P21, P30, P60, P90, P120, P180). The equal volumes of retrograde fluorescent tracer FluoroGold (FG) were injected into the selected regions of the motor or somatosensory cortices. The volume of the dorsal part of the claustrum, total number of projecting neurons, numerical density and percentage distribution of projecting neurons were estimated by means of the unbiased stereological methods. The claustrocortical connections both with the motor and somatosensory areas in a rabbit are established in the postnatal life. The parts of the claustrum occupied by the motor and somatosensory projection zones as well as the morphology of the cortically projecting neurons do not reveal characteristic changes during the studied period. The significant decrease of the total number and numerical density of cortically projecting neurons as well as the increase of the claustral volume may reflect the process of adjustment of the claustrum to its modulatory function upon corresponding cortical areas. The intensity of the claustral connections with the motor and somatosensory cortices reveals significant difference during the studied period, being higher for the motor projection. It may be assumed that the claustrocortical connections established before birth undergo significant quantitative changes during postnatal development.

The connections of the endopiriform nucleus with the insular claustrum in the rat and rabbit.

The connections between two parts of the claustrum in the rat and rabbit were studied using the highly fluorescent lipophilic carbocyanine dye (Dil). After the application of Dil crystal into the endopiriform nucleus, labeled fibers in the insular claustrum were observed in its part directly neighboring the insular cortex and capsula externa. Additionally, numerous projections into the piriform, insular and entorhinal cortices were present. The presence of connections between the endopiriform nucleus and insular claustrum suggests its role concerned with the processes taking part in the allocortical regions as well as in the limbic system.

Qualitative and quantitative differences in the motor and somatosensory cortical projections of the rat claustrum--combined retrograde transport and stereological studies.

Using axonal retrograde tracing, combined with morphometric analysis, we compared the distribution and number of claustral neurons projecting to the motor and somatosensory cortical areas in the Wistar rat. Comparable volumes of the retrograde tracer Fluoro-Gold, were injected into the motor or somatosensory cortices. Injections into these areas resulted in labeling of neurons along the entire length of the claustrum. Neurons retrogradely labeled after injection into the motor cortex prevailed in the anterior part of the claustrum, whereas those projecting to the somatosensory cortex predominated in the central part. The mean number of claustral neurons retrogradely labeled after tracer injections into the motor cortex significantly outnumbered that from the somatosensory cortical area (p < 0.01). Similarly, the mean value of the numerical density of the retrogradely labeled neurons was significantly higher for the motor projection zone in the claustrum, than for the somatosensory projection zone (p < 0.001). The contralateral claustral projections, both into the motor and somatosensory cortices, were considerably lower in number than the ipsilateral ones. These findings indicate that: (1) the claustral projections to the various cortical regions seem to be differentiated (2) the distribution of claustral neurons projecting to the motor and somatosensory neocortical areas shows an anteroposterior gradient, (3) the claustrum of the rat appears to be more closely related to the motor than to the somatosensory system, (4) the rat claustrum seems to function more as a satellite than a relay structure in relationship to the cerebral cortex.

Claustrocingulate connections in the rabbit and rat--a stereological study.

Unbiased stereological methods were used for estimating the numerical density and the total number of claustral neurones projecting to the cingulate cortex in rabbit and rat. In rat the numerical density of neurones projecting to the retrosplenial granular cortex (RSG) differed significantly from those projecting to the retrosplenial agranular (RSA) and cingulate (Cg) cortices while in rabbit the numerical densities of retrogradely labelled neurones in the claustrum following injections into various areas of the cerebral cortex did not differ significantly. The total number of retrogradely labelled neurones in the claustral limbic zones did not differ significantly in both species. The quantitative analysis of claustral zones projecting to a different cingulate cortex area, both in rabbit and rat, reveals that each of these zones is rather homogeneous.

The limbic zone of the rabbit and rat claustrum: a study of the claustrocingulate connections based on the retrograde axonal transport of fluorescent tracers.

The claustrum is a subcortical structure lying under the insular and piriform cortices, whose function is still not clear. Although data exist on connections of the claustrum and the limbic cortex, the topography of the limbic zone in the rabbit and rat claustrum has not been studied extensively. The study was performed on 17 adult Wistar rats and 12 New Zealand rabbits. Two percent water solutions of fluorescent retrograde tracers fast blue and nuclear yellow were injected into the various regions of the limbic cortex. The limbic zone is localized throughout the whole rostrocaudal extent of the claustrum, mainly in its ventromedial portion lying close to the external capsule. Although this zone of the claustrum is localized similarly in both rat and rabbit, some differences between these two species exist. In the rat, neurons projecting to all limbic areas are localized mainly in the anterior and central parts of the claustrum, whereas in the rabbit, the majority of the neurons projecting to the cingulate cortex are present in the anterior and central parts of this structure, while neurons sending axons to the retrosplenial cortex are localized in the central and posterior parts. In both species, double-labeling study showed that neurons projecting to various limbic regions are intermingled and that neurons sending axons into two different limbic regions are seen only occasionally. Our findings give support to the role of the claustrum in integrating information between different areas of the cerebral cortex and the limbic system.

The combined retrograde transport and unbiased stereological study of the claustrocortical connections in the rabbit.

The quantitative analysis of the claustrocortical connections in the rabbit, labeled with the fluorescent retrograde tracer Fluoro-Gold (FG), was conducted by means of unbiased stereology. The FG was injected into selected regions of the motor, somatosensory, auditory and visual cortices and then a comparison of the various claustrocortical projections was carried out. This was achieved by comparing (1) the numerical densities of projecting neurones for each claustral projection zone and (2) the distribution of the labeled neurones throughout the rostro-caudal extent of the claustrum. No significant differences between the numerical densities of labeled neurones in the various projection zones are reported. The motor and primary somatosensory projections dominated in the anterior and central parts of the claustrum, whereas the secondary somatosensory, auditory and visual projections--in the posterior part. The difference in the distributions was significant (p < 0.001). Summarizing, the cortical projections in the claustrum, although varying topographically, do not reveal a quantitative differentiation. This may speak in favour of the integrative and modulating function of this structure in relationship to the neocortex.

The piriform cortex and the endopiriform nucleus in the rat reveal generally similar pattern of connections.

The afferent and efferent connections of the piriform cortex and the endopiriform nucleus in the rat were studied by the method of axonal transport of two fluorescent tracers: Fluoro-Gold and Fluoro-Ruby. The results indicate that both structures possess not only the connections with the olfactory system, but also the reciprocal connections with the limbic system (entorhinal cortex, amygdaloid body), thalamus (mediodorsal and midline nuclei), extrapyramidal system (ventral part of the nucleus accumbens). The topographic differences in the organization of the association connections between the anterior and posterior parts of the piriform cortex are reported. Additionally, the reciprocal and relatively numerous connections between the endopiriform nucleus and the piriform cortex may result in their modulatory function, which in some pathological circumstances may have a critical significance in epileptogenesis.