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.

The shape of the inferior horn of the lateral ventricle in relation to collateral and occipitotemporal sulci.

The shape of the inferior horn of the lateral ventricle has been investigated in 100 (50 right and 50 left) human brain hemispheres which were fixed in 40% formaldehyde solution and cut frontally. It has been found that the shape of the inferior horn depends on the course and depth of the collateral and occipitotemporal sulci. In most cases a part from two main ventricular surfaces: the hippocampal and superolateral, there is one surface more, either the inferior-collateral eminence (Type I--97% of cases in the posterior part of the inferior horn, 49%--in the middle and 42%--in the anterior part) or the inferolateral, adjecent to the occipitotemporal sulcus (Type II--0%, 26% and 35%, respectively). In a few cases both collateral eminence and inferolateral surface were present (Type III--3%, 2% and 6% respectively). In type IV neither collateral eminence nor the inferolateral surface appeared (0%, 23%, 17% respectively). The authors suggest to designate the inferolateral surface, not mentioned in the literature, as the occpitotemporal eminence (of the inferior horn).

Visual zone of the claustrum shows localizational and organizational differences among rat, guinea pig, rabbit and cat.

The retrograde axonal transport method was used to compare the topography and organization of the visual zone of the claustrum in rat, guinea pig, rabbit and cat. First, massive Fluoro-Gold injections were placed into the primary visual cortex and the secondary areas. Experiments showed differences in the location of the visual zone among the animals under study. In rat, the visual zone occupied the posteroventral part of the claustrum and spread to its anterior pole. In guinea pig, neurons projecting to the visual cortex were located dorsally in the posterior half of the claustrum. In rabbit, similarly to the rat, they were localized in the posteroventral part; however, they did not reach the anterior pole. In cat, neurons that project to the visual cortex were concentrated dorsally in the posterior fourth of the claustrum. In double-injection experiments, Fast Blue and Diamidino Yellow were placed into the primary and secondary visual areas in various combinations. The experiments showed that in the rat and the rabbit claustral neurons project to primary visual cortex (area 17) as well as to both secondary visual areas (areas 18a and b). Populations of neurons sending axons to the primary and secondary areas showed full overlap. The presence of double-labeled neurons indicates that some claustral neurons project both to the primary and secondary fields. In cat, neurons that project to the primary visual cortex appear to be clearly separated from those connected with the secondary visual area, as no double-labeled neurons were found. In all studied species, the double injections placed into the visual and primary somatosensory cortex did not result in any double-labeling neurons. Our results indicate that the location of the visual zone in the posterior part of the claustrum is a phylogenetically stable feature, whereas its dorsoventral shift as well as the extent toward the anterior pole is related to the particular species. The overlap of neurons projecting to the primary and secondary visual areas in the rat and rabbit as well as the separation of both projections in cat appear to reflect the higher degree of complexity of the visual system in the latter.

The cortico-related zones of the rabbit claustrum-study of the claustrocortical connections based on the retrograde axonal transport of fluorescent tracers.

The claustrocortical connections in the rabbit were assessed for the first time by the method of axonal retrograde transport of two fluorescent tracers (Fast Blue and Diamidino Yellow). The material consisted of 23 adult New Zealand rabbits. Projection zones of spindle-like form, connected with the precentral, postcentral, temporal and occipital cortices have been delineated. They are organized topographically both in the anteroposterior and ventrodorsal direction. The precentral (motor) projection zone is localized in the anterodorsal part of the claustrum. It may be divided into two separate parts that project to the medial and lateral part of the precentral cortex. The large postcentral (somatosensory) zone occupies mainly the central part, whereas the temporal (auditory) and occipital (visual) zones are situated in the posteroventral part of the claustrum. The overlap of various claustral projection zones is differentiated, the largest being that of the somatosensory zones. In comparison to the results of study of claustral projection zones performed on other species, presumably on the rat and cat, its seems plausible to conclude that the extension of claustral projection zones and degree of their overlap in the rabbit represent an intermediate character.

Ultrastructural organization of the visual zone in the claustrum of the cat.

Data obtained by using ultrastructural and morphometric approaches revealed three types of neurons in the cat visual claustrum. The most numerous were medium-sized and large ones. They formed 3/4 of the cell population. The ultrastructural properties of those cell types were largely similar. Their cell bodies were oval, round, fusiform or triangular and contained more or less indented nuclear envelope. The cytoplasm of those cells was characterized by a high concentration of subcellular organelles and particularly rough endoplasmic reticulum. The characteristic feature of those cells was a low nucleus/cell body area ratio (47 +/- 1% and 43 +/- 1%, respectively). The proximal dendrites of medium-sized cells were usually wide at the base, relatively short and tapering, whereas, those arising in the large cells were often thick and had a short tapering base. The neurons described above stained by Golgi impregnation method showed spines on their distal dendrites both under the light and electron microscopy. The retrograde axonal transport of HRP and WGA-HRP following injections into the visual cortex confirm that they are mainly projection cells which form the ascending limb of the claustrocortical loop. The third type of neurons formed a less numerous group of small cells which differed from the larger ones in various respects. They possessed the large nuclei with deeply indented nuclear envelope and comparatively a thim layer of cytoplasm poor in subcellular organells among which free ribosomes and mitochondria were common. The nucleus/cells body area ratio high (59 +/- 2%). In Golgi preparations their dendrites did not show spines. The dendrites originating from that type of neurons were thin, long and did not posses a wide tapering base. They are mainly claustral intrinsic neurons.

Rat's claustrum shows two main cortico-related zones.

Methods of retrograde axonal transport were employed to evaluate the topography and overlap of claustroneocortical connections in the rat. Fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) were injected simultaneously in various combinations into the motor, somatosensory, auditory and visual cortical areas. Experiments showed that claustroneocortical projections are organized in two main cortico-related zones: sensorimotor and visuoauditory. The sensorimotor zone occupies the anterodorsal part whereas the visuoauditory occupies the posteroventral part of the claustrum. Between these two main zones only a scanty overlap was observed. In the sensorimotor zone a large overlap between neurons projecting to the motor and somatosensory cortical areas exists. The visuoauditory zone is characterized by a full overlap of neuronal populations projecting to the visual and auditory areas.

Some claustral neurons projecting to various neocortical areas show morphological differences.

The morphology of claustral neurons projecting to the motor, somatosensory, auditory and visual cortical areas in the rat was analyzed by means of combination of axonal retrograde transport and morphometric analysis. Fluoro-Gold (FG) injections placed into various cortical fields resulted in labeling in the claustrum four neuronal types: pyramidal with thick main dendrite, oval with a few thin dendrites spreading out in various directions, fusiform possessing two main dendrites arising from opposite poles of the cell body and polygonal. Pyramidal neurons prevailed in populations of neurons projecting to the motor cortex of the contralateral hemisphere. Oval neurons outnumbered other types in populations projecting to the somatosensory, auditory and visual cortical fields. The number of fusiform and polygonal neurons did not exceed at 12.5% together in any populations. Neurons projecting to the contralateral hemisphere were the largest claustral neurons (mean cross-section are 167.19 +/- 2.9 micron 2) whereas neurons projecting to the motor cortex where the largest claustral neurons projecting ipsilaterally (141.89 +/- 2.22 micron 2). There was no significant difference between neurons projecting to the somatosensory (113.46 +/- 1.9 micron 2) cortex and to the visual (111.8 +/- 1.4 micron 2) cortex whereas neurons related to the auditory are (95.98 +/- 1.75 micron 2) were the smallest claustral neurons. These observations pointed out that the morphology of claustral neurons is closely related to a cortical area to which they send axons.

Acetylcholinesterase activity as a marker of maturation of the basolateral complex of the amygdaloid body in the rat.

Development and maturation of the basolateral complex of the amygdaloid body were studied in fetal and postnatal rat brains. In cresyl violet-stained sections the basolateral complex was distinguishable at the 17th prenatal day. On about the 14th postnatal day, it showed all the features of the adult structure. The acetylcholinesterase activity appeared in neuropil of the basolateral complex on the seventh postnatal day; it increased slowly up to the 60th day. After that age, the acetylcholinesterase activity was stable in all parts of the basolateral complex and corresponded to its cytoarchitectonic differentiation. Our results suggest that the process of maturation of the amygdaloid basolateral complex, being related to the ingrowing of the cholinergic fibers from the basal forebrain, lasts at least up to the end of the second month of postnatal life.

Alzheimer's disease severely affects areas of the claustrum connected with the entorhinal cortex.

Pathological changes in the claustrum and its main parts (dorsal, temporal, orbital and paramygdalar) were studied on ten brains of patients with Alzheimer's disease and five control brains. The brains after fixation in formalin were embedded in paraffin, coronal-8-micron-thick serial sections were cut and stained either with cresyl violet or with immunocytochemical methods for amyloid and tangles. Morphometrical studies were performed in all parts of the claustrum along its whole extension. The claustrum in control brains was free of neurofibrillary and amyloid pathology except for one case with few senile plaques in the paramygdalar part. In AD affected brains the most severe changes were found in the paramygdalar part connected with the entorhinal cortex (neuronal loss -46%; 698 +/- 244.6 neurofibrillary tangles per mm3, tangle/neuron ratio -6.8 +/- 2.4%). In other parts of the claustrum related mainly with the neocortex pathological changes were significantly less expressed. As pathological AD type alteration affects severely practically the whole hippocampal formation, including the entorhinal cortex, the extensive neuronal pathology of the paramygdalar part of the claustrum is probably related to this process and may deeper memory dysfunction.

Division of the human claustrum according to its architectonics and morphometric parameters.

The topography and cytoarchitectonics of the claustrum as well as morphometric parameters of its neurons were studied in 10 human brains obtained from patients without any detectable neuropathological changes. We distinguished four parts of the claustrum: dorsal, orbital, temporal and paraamygdalar. The dorsal and orbital parts contain larger cells, than those of the temporal and paraamygdalar parts, although these differences were statistically non significant. The highest neuronal density was observed in the paraamygdalar part. The nucleus and nucleus@cell body area ratio was significantly smaller in the dorsal part than in other parts of the claustrum. We described three types of neurons in the claustrum: (1) medium-sized either fusiform or triangular cells with darkly stained cytoplasm; they predominate in the dorsal and temporal parts, (2) medium-sized as well as large cells, either multipolar or pyramidal-like with lightly stained cytoplasm; they are most numerous in the orbital and paraamygdalar parts, (3) small, multipolar or oval neurons with darkly stained ring of cytoplasm; these types of neurons are uniformly distributed throughout all parts of the claustrum. The subdivision of the human claustrum is in accordance with our observations that each of these parts possesses connections with different cortical regions.

Amyloid deposits and loss of neurones in the claustrum of the aged dog.

In dogs under 17 years of age no amyloid deposits were observed in the claustrum. After that age amyloid deposits were found in all the brains studied. The number of amyloid deposits increases rapidly between 17 and 19 years of age. In none of the animals were neurofibrillary tangles found. However, in almost all claustra with amyloid deposits the number of neurones was smaller than in those without amyloid pathology. The loss of neurones resulted in a decrease in the volume of claustrum, which is about 22% smaller in dogs over 17 years of age than in younger animals. No considerable age-related differences were found in the average size of neurones and their nuclei.

Insular claustrum of the dog--a morphometric investigation of cellular structure.

Investigation of the dog's insular claustrum was performed using morphometric methods. The brains of 14 dogs (7-14 year-old) were studied. The size, shape and density of neurons are different in various parts of the insular claustrum. The posterior part, related to the visual system, is heterogenous and possesses medium-sized and small densely packed neurons, the average cross-sectional area of cell body being 175.8 +/- 45.5 micron2. The similar average cross-sectional area of cell body (174.1 +/- 55.3 micron2) was found in the central part of the insular claustrum. However, neuronal architecture is here less heterogenous and neurons are less densely arranged. The anterior part, connected mainly with the frontal and motor cortex, is more homogenous and contains loosely arranged neurons of a rather large size (average size--198.7 +/- 53.46 micron2).

Morphometric parameters of some hypothalamic nuclei: age-related changes.

The morphometric analysis of some hypothalamic nuclei was performed on 20 brains of subjects of both sexes aged from 29 to 79 years. They were divided into two groups: below and above the age of 60. Supraoptic (SON), lateral tuberal (LTN) and lateral mammillary (LMN) nuclei were investigated. In the group of aged subjects a decrease in cross-section area of neuron nuclei was observed in all investigated structures, expressed most in the LTN. A considerable increase in the neuron density with age was found in the LTN. The size of neuron cell bodies and the number of neurons did not change significantly with age in any of the investigated nuclei.

Lateral tuberal nucleus in man and macaca comparative morphometric investigations.

Cytoarchitectonics of the lateral tuberal nucleus--a phylogenetically new hypothalamic structure--was investigated using morphometric methods. The most characteristic feature of the human lateral tuberal nucleus is an extremely great variability of its shape and segmentation. The lateral tuberal nucleus consists mostly of several separated neuronal groups; in each brain the arrangement of these groups is different. In macaca the lateral tuberal nucleus is a single elongated neuronal structure much less variable than in man. The human LTN is approximately ten times larger than that in macaca, but the number of neurons is only about four times higher due to a lower neuronal density. The lateral tuberal nucleus both of man and macaca is a homogenous neuronal population with a very low coefficient of variability of cross-section neuronal area (30% and 24% respectively). The size of neuronal bodies in the lateral tuberal nucleus is about 42% smaller in macaca than in man. However, the size of neuronal nuclei is slightly (15%) bigger and the nucleus/cell body area ratio is about twice as high in macaca as that in man (40% and 21% respectively).

Relationship of corticoclaustral axon terminals to neurons of the claustrum in the cat.

A dark type degeneration of axon terminals was observed in the dorsocaudal part of the claustrum following lesions of the visual cortex. Most of the degenerating axon terminals had synapses mainly with dendritic spines (64.1%) and small dendrites (25.1%). Only 5.8% of degenerating axon terminals formed synaptic contacts with medium-sized dendrites, 1.0% with large dendrites and 0.5%--with other parts of the claustral neurons. 3.5% of degenerating axon terminals were observed to form a contact with more than one postsynaptic structure. Degenerating axon terminals displayed synaptic contacts exclusively of asymmetrical type with clearly identifiable postsynaptic densities which suggest corticoclaustral afferents to be excitatory. Our data strongly support the notion that there are direct synaptic contacts between the ascending arm of the corticoclaustral neuronal loop.

Variability of the parahippocampal surface of the transverse fissure in the human brain.

The parahippocampal (ventral) surface of the lateral part of the transverse fissure (LTF), formed by the parahippocampal gyrus, was examined in 53 human brain hemispheres without pathological changes. Cytoarchitectonics of this region was studied on the frontal histological sections stained either with cresyl violet or with Weil method. Four types of the shape of the parahippocampal surface of LTF were distinguished. In type 1 the surface is either flat or elevated in its medial part (medial eminence; 34.0%). In type 2 the surface is elevated in its lateral part (lateral eminence, 24.5%). In type 3 there are two eminences--the lateral and the medial; both of similar height (20.8%). In type 4 the lateral eminence is distinctly higher than the medial one (20.7%). The eminences contain cytoarchitectonically different structures. The lateral eminence is formed mainly by the subiculum and CA1 area, the medial by the presubicular region. The shape of the parahippocampal surface of LTF may be of importance in assessment of the Alzheimer's disease pathology.

The second layer neurones of the entorhinal cortex and the perforant path in physiological ageing and Alzheimer's disease.

The hippocampal formation was studied in 5 brains of younger (29 to 52 years of age) and 6 brains of elderly (61 to 89 years of age) subjects without signs of dementia, as well as in 11 brains of patients with Alzheimer's disease (65 to 91 years of age). The 8-microns-thick sections were stained either with cresyl violet, Weil method or with immunocytochemical methods for amyloid (4G8) and neurofibrillary tangles (Tau-1). Cell bodies, senile plaques and tangles were counted in all brains. In brains of patients with Alzheimer's disease a significant neuronal loss (about 56%) was observed in the second layer of the entorhinal cortex. The tangles/neurones ratio was very high (62.79.1%) in this layer. A great number of senile plaques were present in the whole hippocampal formation, especially in the molecular layer of the dentate gyrus (22.91.5 plaques/mm2) which is the termination zone of the perforant path. It seems therefore, that pathological alterations in Alzheimer's disease disrupt the main input to the hippocampal formation. In "physiological" ageing we did not observe changes in the density of neurones, although single tangles and plaques were found in all hippocampal areas. In elderly individuals 3.81.3% of neurones of the second layer revealed neurofibrillary pathology; a few plaques were found in various areas of the hippocampal formation. These observations may suggest only a slight decrease in number of neurones in the hippocampal formation. However, these changes cause a slight impairment of memory and learning often found in elderly individuals without dementia.