Human spermatozoa antigens in unexplained infertility.

OBJECTIVE: To determine and compare the immunolocalization of functionally important antigens in human spermatozoa in an unexplained infertility (UI) group. MATERIALS AND METHODS: In this study, the sperm samples of 20 patients undergoing evaluation belonging to normozoospermic group, whose primary reason of infertility was under investigation for this purpose, were screened. CD46, CD55 and CD52, CD69, CD98, fMLP, HI307, and 80280 were stained on the spermatozoa through indirect immunofluorescence technique. RESULTS: In addition to CD46, CD55, and CD52 antigens, which are known to be localized on human spermatozoa, significant immunolocalization of several novel antigens including: CD52, CD69, CD98, fMLP, HI307, and 80280 were determined on the spermatozoa of the unexplained infertility group, possibly reflecting important roles in the pathophysiology of such unresolved clinical situations. CONCLUSION: Identification and characterization of antigens present on sperm cells is crucial for understanding of the diagnosis and treatment of unexplained infertility. Further studies were conducted to evaluate a possible correlation between the expression of these antigens and clinical outcomes in different well-defined infertility groups.

Effect of intra-articular injection of levobupivacaine on articular cartilage and synovium in rats.

OBJECTIVE: Intra-articular local anesthetics are often used for prevention of pain after arthroscopic knee surgery. However, the effect of local anesthetics other than bupivacaine on articular cartilage and synovium has not been studied. Also, complications associated with the injection of intra-articular bupivacaine have appeared in the literature. The aim of this study was to evaluate the effects of levobupivacaine on the articular cartilage and the synovium in rats. METHODS: Under aseptic conditions 0.25 ml (5 mg/ml) of levobupivacaine was injected into the right knee joint while 0.25 ml of saline was simultaneously injected into the left knee joint of 20 adult Sprague-Dawley rats. The purpose of saline injections was to serve as a control group. Groups of five rats were killed on days 1, 7, 14 and 21 after administration of injections. The knee joint samples were evaluated for the presence of inflammation in the articular and periarticular tissues and the synovium. RESULTS: There were no significant differences between the levobupivacaine and control groups with respect to inflammation in the articular and periarticular tissues and the synovium. CONCLUSIONS: Although more studies are needed before final recommendations can be made, by evaluating the results obtained from this study, the clinical use of intra-articular levobupivacaine can be recommended for arthroscopic knee surgery.

Comparison of reflection contrast microscopy and electron microscopy on the histopathological diagnosis of various kidney diseases.

Our aim in this study was to compare reflection contrast microscopy (RCM) with transmission electron microscopy (TEM) to understand whether RCM could be used in the histopathological diagnosis of various kidney diseases as a less expensive and an easier alternative to TEM. The diagnoses of kidney pathologic lesions included Alport syndrome, thin membrane disease, Ig A nephropathy. RCM is a form of light microscope that works in the reflected mode, suitable to observe ultrathin (50-100 nm) plastic sections that is also used in TEM. Our findings showed that RCM showed similar results compared with TEM on these lesions described earlier.

Ciliary neurotrophic factor-immunoreactivity in olfactory sensory neurons.

Ciliary neurotrophic factor (CNTF) has been implicated in processes of neuroprotection, axonal regeneration and synaptogenesis in the lesioned CNS. In the olfactory system, which is characterized by particularly robust neuroplasticity throughout life, the concentration of CNTF is high even under physiological conditions. In the present study, the cellular localization of CNTF-immunoreactivity was studied in the rat and mouse olfactory epithelium. In both species, individual olfactory sensory neurons (ONs) displayed intense CNTF-immunoreactivity. The number of CNTF-ir ONs varied interindividually in rats and was lower in mice than in rats. In olfactory epithelia of mice expressing beta-galactosidase under control of the CNTF promoter, cells of the ON layer were immunoreactive for the reporter protein. CNTF-ir ONs were olfactory marker protein-positive and growth associated protein 43-negative. CNTF-ir ONs lacked apoptotic markers, and the number of specifically labeled ONs was apparently unchanged after light chemical lesioning of the epithelium, indicating that CNTF-immunoreactivity was not associated with ON death. Electron microscopy of CNTF-ir ON axons in innervated olfactory bulb glomeruli documented that they formed typical ON axonal synapses with target neurons. Three dimensional reconstructions of bulb pairs showed a striking similarity of the positions of glomeruli innervated by CNTF-ir ON axons in left and right bulbs of individual animals and interindividually. The number of innervated glomeruli differed interindividually in rats and was lower in mice than in rats. The results show that in rodents CNTF-immunoreactivity occurs in a subset of mature, functionally competent ONs. The localization of target glomeruli suggests that CNTF-immunoreactivity may be associated with the expression and/or activation of specific olfactory receptor proteins.

The corticotropin-releasing factor (CRF)-system and monoaminergic afferents in the central amygdala: investigations in different mouse strains and comparison with the rat.

Corticotropin-releasing-factor (CRF) containing systems and monoaminergic afferents of the central amygdaloid nucleus (Ce) are crucial players in central nervous stress responses. For functional analyses of specific roles of these systems, numerous mouse models have been generated which lack or overexpress individual signal transduction components. Since data concerning system morphologies in murine brain are rarely available, mouse studies are usually designed and interpreted based on previous findings in rats, although interspecies differences are frequent. In the present study, in situ hybridization for CRF mRNA and correlative immunocytochemistry for CRF and monoaminergic afferents revealed numerous CRF mRNA-reactive neurons in the lateral Ce subnucleus (CeL) codistributed with dense dopaminergic fiber plexus in mice as has been demonstrated in rats. However, while in rats the lateral capsular Ce (CeLc) displays only scarce CRF immunoreactive (CRF-ir) innervation, particularly dense CRF-ir fiber plexus were observed in the CeLc in mice, with differences in labeling densities between different strains. CRF-ir terminal fibers overlap with the moderate serotonergic innervation of this subnucleus in mice. Additionally, CRF mRNA-reactive neurons were found immediately dorsal to the amygdala in the region of the interstitial nucleus of the posterior limb of the anterior commissure/amygdalostriatal transition area in both species. In mice, this region displayed dense CRF-ir fiber plexus, with variations between the strains. The results indicate that in mice and rats dopaminergic afferents represent the primary monoaminergic input to the CRF neurons in the CeL. In mice only, CRF-ir afferents provide dense innervation of CeLc neurons. Since the CeLc lacks dopaminergic input in both species but possesses moderate serotonergic afferents, CRF/serotonin interactions may occur selectively in mouse CeLc. The observed interspecies and interstrain differences in CRF input and CRF/monoaminergic interactions may influence the interpretation of findings concerning Ce functions in stress and fear in mouse models.

Quantitation of catecholamine neurons in the locus coeruleus in human brains of normal young and older adults and in depression.

A quantitative study of the morphology and distribution of norepinephrinergic neurons in the human locus coeruleus (LC) is given for normal young and older adult brain. Norepinephrine (NE)-producing neurons are identified by immunocytochemistry of two NE biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), visualized by the peroxidase-antiperoxidase and immunogold-silver-staining methods. TH and DBH immunoreactions yield equivalent results. Both immunocytochemical visualization methods allow detailed analysis of neuronal morphology. The neurons of the human LC fall into four classes: large multipolar neurons with round or multiangular somata, large elliptical "bipolar" neurons, small multipolar neurons, and small ovoid "bipolar" neurons. Though most of the neurons contain neuromelanin pigment, some larger neurons lack pigmentation. Dendritic arborization of all neurons is extensive. Computer-assisted quantitative measurements of the parameters somatic size, dendritic arbor length, surface area, and volume are given. Somatic areas of LC neurons of all four classes are decreased in older adult brain, but dendritic arborization is equally extensive as in the younger. The rostrocaudal length of the LC is approximately 15 mm, and no age-dependent decrease is observed. Computer-assisted mapping of immunoreactive neurons and three-dimensional reconstruction allow division of the LC into rostral, middle, and caudal parts with characteristic distribution of neurons. Small neurons predominate in all parts, but the relative contribution of larger cells decreases in a rostrocaudal direction. A cell loss of 27-37% occurs in older adult brains and to 55% in the brain of a chronically depressed patient without dementia. Cell loss is highest in the rostral part, lower in the middle, and absent in the caudal part, and more small cells are lost than larger ones.

Alterations in catecholamine neurons of the locus coeruleus in senile dementia of the Alzheimer type and in Parkinson's disease with and without dementia and depression.

The present study provides qualitative and quantitative investigations of the norepinephrine (NE) neurons in the locus coeruleus (LC) in two neurodegenerative disorders, the senile dementia of the Alzheimer type (SDAT) and Parkinson's disease (PD). The group of PD subjects was subdivided into cases without dementia (P - D), cases with dementia, L-dopa responsive (P + D), and cases with fulminant dementia whose motor disorder symptoms were L-dopa nonresponsive (P + D/L-dopa non-responsive). NE neurons were demonstrated by immunocytochemistry against tyrosine hydroxylase (TH). Quantitations of neuronal parameters and cell numbers and three-dimensional reconstructions of the LC were carried out with a computer-assisted system. In SDAT cases, the rostrocaudal LC length (13 +/- 2.2 mm) is shorter than in controls (14.9 +/- 1.4 mm). The four basic LC neuron classes found in the normal human brain (large multipolar, large "bipolar," small multipolar, and small "bipolar" neurons; Chan-Palay and Asan: J. Comp. Neurol. this issue) are recognizable, but many cell somata are swollen and misshapen with fore-shortened, thick, and less branched dendrites. LC neuron numbers are reduced (between -3.5% and -87.5%). Neuron loss is greatest in the rostral part, less in the middle, and least in the caudal part. In PD cases, the rostrocaudal length (12.4 +/- 1.5 mm) is shorter than in SDAT and controls. The neuronal morphology is more severely altered than in SDAT. The basic neuron classes are hardly distinguishable. Most cell bodies are swollen; they frequently contain Lewy bodies; and the dendrites are short and thin with absent or reduced arborizations. Neuron numbers are more reduced than in SDAT (between -26.4% and -94.4%). Alterations are as severe caudally as rostrally in P - D, and P + D/L-dopa nonresponsive cases. P + D cases are more severely affected rostrally. The presence of depression in SDAT and Parkinson's patients is accompanied by the greatest loss of LC neurons. On the basis of morphological alterations of the TH-immunoreactive neurons, and the degree and topographical distribution of neuron loss, a differentiation is possible between the LC in normal brain and that in SDAT and PD for diagnostic purposes.

Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry.

Non-radioactive in situ hybridization using complementary RNA and oligonucleotide probes was applied in order to clearly identify the cell types expressing GLT1 and to show their regional distribution in the central nervous system of the rat. The results were compared with immunocytochemical data achieved using an antibody against a synthetic GLT1 peptide. The study showed that GLT1 was expressed in astrocytes and Bergmann glia which were identified by the detection of an astrocytic marker protein. Additionally, subsets of neurons in different brain regions (e.g., CA3/4 pyramidal cells of the hippocampus, endopiriform nucleus) were labelled by in situ hybridization. In other cell types of the central nervous system (oligodendrocytes, ependymal cells, epithelal cells of the choroid plexus, tanycytes), GLT1 expression was not detectable. The generally dense astrocytic immunolabelling of the gray matter of the brain showed an even higher intensity in regions reported to show high glutamatergic activity and astrocytic glutamate metabolism (e.g., the termination field of the glutamatergic perforant path in the hippocampus). On the basis of the cellular regional distribution of the GLT1 messenger RNA and protein demonstrated in the present study, it is reasonable to assume that this high affinity transporter is of importance for the maintenance of adequate extraneuronal glutamate levels.

A splice variant of glutamate transporter GLT1/EAAT2 expressed in neurons: cloning and localization in rat nervous system.

Rapid uptake of synaptically released glutamate via the high affinity glutamate transporter 1 (GLT1; EAAT2) is important for limiting transmitter signalling and prevents a harmful receptor overstimulation. So far, in the adult brain GLT1 protein has only been detected in astrocytes. Here, we describe the cDNA cloning of a variant of GLT1 from rat brain which is generated by alternative splicing at the 3'-end of the GLT1 cDNA. Reverse transcription-polymerase chain reaction revealed that the GLT1 variant message was present not only in brain, but also in peripheral organs. Northern blot analysis showed that in brain the mRNA of GLT1 (approximately 11 kb) is predominant while in the retina the mRNA of GLT1 variant (approximately 12.5 kb) prevails. In situ hybridization using cRNA and oligonucleotide probes, and immunocytochemistry using an antibody against a synthetic GLT1v peptide were applied in order to identify the cell types expressing GLT1 variant in the adult rat nervous system. GLT1 variant is preferentially expressed in neurons of the CNS and PNS, but is also detected in glial cells (oligodendrocytes, ependymal cells, epithelial cells of the plexus choroideus, satellite cells, and Schwann cells). In contrast to GLT1, GLT1 variant was only occasionally detected in astrocytes. Immunolabelling revealed a preferentially cytoplasmic (frequently granular) staining of neurons and glial cells, suggesting a localization of GLT1 variant protein in vesicle membranes. The studies provide evidence that the cellular expression of the GLT1 variant in the CNS is almost complementary to that of GLT1 and that the GLT1 variant does not seem to be restricted to the CNS.

Ciliary neurotrophic factor in the olfactory bulb of rats and mice.

Ciliary neurotrophic factor (CNTF) is primarily regarded as an astrocytic lesion factor, promoting neuronal survival and influencing plasticity processes in deafferented areas of the CNS. Postnatal loss of neurons in CNTF-deficient mice indicates a function of the factor also under physiological conditions. In the olfactory bulb, where neurogenesis, axo- and synaptogenesis continue throughout life, CNTF content is constitutively high. The cellular localization of CNTF in the rat olfactory bulb is not fully resolved, and species differences between mouse and rat are not yet characterized. In the present study, four different CNTF antibodies and double immunolabeling with specific markers for glial and neuronal cells were used to study the cellular localization of CNTF in rat and mouse olfactory bulb. Specificity of the detection was checked with tissue from CNTF-deficient mice, and investigations were complemented by immunolocalization of reporter protein in mice synthesizing beta-galactosidase under control of the CNTF promoter (CNTF lacZ-knock-in mice). In both species, CNTF localized to ensheathing cell nuclei, cell bodies and axon-enveloping processes. Additionally, individual axons of olfactory neurons were CNTF immunoreactive. Both CNTF protein content and immunoreaction intensity were lower in mice than in rats. Scattered lightly CNTF-reactive cells were found in the granular and external plexiform layers in rats. Some CNTF-positive cells were associated with immunoreactivity for the polysialylated form of the neural cell adhesion molecule, which is expressed by maturing interneurons derived from the rostral migratory stream. In CNTF lacZ-knock-in mice, beta-galactosidase reactivity was found in ensheathing cells of the olfactory nerve layer, and in cells of the glomerular, external plexiform and granular layers. The study proves that CNTF is localized in glial and neuronal structures in the rodent olfactory bulb. Results in mice provide a basis for investigations concerning the effects of a lack of the factor in CNTF-deficient mice.

Localization of cGMP-dependent protein kinase type II in rat brain.

In brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP-dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP effects in brain requiring protein phosphorylation.

The catecholaminergic innervation of the rat amygdala.

The present study is the first to demonstrate conclusively and to analyze systematically synaptic contacts of all three types of catecholaminergic afferent fibers in different nuclei of the rat amygdala and to relate the catecholaminergic innervation to neurochemically identified target neurons. 4.1.1 Central Nucleus: The central nucleus is the amygdaloid nucleus receiving the most dense catecholaminergic innervation. In the medial central nucleus, dopaminergic, noradrenergic and adrenergic terminal plexus overlap, in the central lateral central nucleus mainly dopaminergic plexus are found. The lateral capsular central nucleus is generally scarcely innervated, but individual neurons of this subnucleus possess a dense dopaminergic innervation. Colocalization of neurotensin in dopaminergic afferents is rare, the majority of the dense neurotensin-ir terminal plexus consist of non-dopaminergic fibers. The catecholaminergic innervation of the medial central nucleus is directed preferentially at peripheral neuronal structures, and has thus presumably modulatory functions. Dopaminergic terminals form predominantly symmetric, noradrenergic and adrenergic terminals from preferentially asymmetric synapses. A characteristic feature of the dopaminergic innervation is the dense perisomatic innervation of selected neurons. Adrenergic and the majority of noradrenergic afferent fibers to the medial central nucleus originate from cell groups in the medulla oblongata and contain high levels of NPY. GAD mRNA-detection suggests that most target neurons of catecholaminergic afferent fibers are capable of synthesizing GABA in the medial central nucleus. In its dorsal part, GABA is possibly colocalized with somatostatin, and many neurons express the dopamine-1-receptor subtype mRNA. In the posteroventral medial central nucleus, on the other hand, enkephalin mRNA-r and dopamine-2-receptor subtype mRNA-reactive neurons show a similar distribution as the GAD mRNA-reactive ones. Contacts could be shown between dopaminergic, noradrenergic and adrenergic axons and NPY- and somatostatin-immunoreactive neurons which are supposedly among the brainstem projection neurons of the medial central nucleus. The dopaminergic innervation of the central lateral central nucleus resembles that of the neighboring striatum in many respects. The synaptic density is high. As in the medial subnucleus, distal neuronal elements are the preferential target structures, indicating a modulatory function possibly regulating the selectivity of the target neurons for stimuli transmitted by other afferent fibers. Besides, individual neurons possess a dense perisomatic, presumably non-selective dopaminergic innervation. The innervation does not appear to be targeted at one specific neurochemical type of neuron in the central lateral central nucleus, but rather contacts somatostatin- and neurotensin-immunoreactive neurons (which are possibly also GABAergic), in addition to GABA/enkephalin-synthesizing and other (e.g., CHAT-immunoreactive) neurons. Individual neurons of the central lateral central nucleus express the dopamine-2-receptor subtype mRNA. The dopaminergic fiber baskets of the lateral capsular central nucleus are found surrounding enkephalin mRNA-reactive neurons. Codistribution studies suggest that they express the dopamine-2-receptor subtype mRNA. 4.1.2 Basal Complex: The basal complex receives dopaminergic and noradrenergic innervation, the latter mainly originating in the locus coeruleus. Some of the dopaminergic afferents contain neurotensin, and in contrast to the central nucleus, all neurotensin-immunoreactive afferent fibers are dopaminergic. In the noradrenergic afferent fibers NPY is not detectable. These results and the innervation pattern displaying mostly peripheral neuronal target structures resemble dopaminergic and noradrenergic innervation patterns documented in cortical areas. (ABSTRACT TRUNCATED)

The adrenergic innervation of the rat central amygdaloid nucleus: a light and electron microscopic immunocytochemical study using phenylethanolamine N-methyltransferase as a marker.

Using immunocytochemistry of phenylethanolamine N-methyltransferase for light and electron microscopy, investigations were carried out to document the morphology of adrenergic afferents innervating the rat central amygdaloid nucleus and to analyse the manner in which contacts with neurons of the nucleus are formed. With the light microscope, dense terminal plexus of phenylethanolamine N-methyltransferase-immunoreactive axons with typical large boutons (diameter > 1 micron) were found in the medial central nucleus, extending into its ventral subdivision and the adjacent intra-amygdaloid portion of the bed nucleus of the stria terminalis. Electron microscopy of the medial central nucleus showed phenylethanolamine N-methyltransferase-immunoreaction product in the cytoplasm of intervaricose axons and boutons. Large adrenergic boutons contained numerous small clear vesicles and, occasionally, large dense-cored vesicles. In serial sections, most boutons formed synaptic contacts. Synapses of immunoreactive terminals were mainly of the asymmetric type and localized preferentially on medium sized to small dendrites and dendritic spines. Structures postsynaptic to adrenergic boutons were often additionally contacted by non-labelled terminals. The study gives evidence that adrenergic afferents exert a direct synaptic influence on medial central nucleus neurons. The peripheral localization of the majority of adrenergic synapses, their asymmetric configuration, and the presence of non-adrenergic synapsing terminals in their immediate vicinity indicate that the major component of the adrenergic input is of an excitatory nature, and is integrated with innervation from other sources.

Histopathological evaluation of the dental pulps in crown-fractured teeth.

Trauma is a common cause of pulpal damage. In traumatic injuries, the first priority is to protect the vitality of pulps. But the time between the trauma and treatment must be short to preserve vital, noninflamed pulps. The aim of this study was to investigate the histopathological changes in pulpal tissues at different time periods after crown fractures. Twenty-three teeth with enamel and dentin fractures, with and without pulp exposure were evaluated. The reasons for seeking dental treatment were aesthetic consideration, pain, or discomfort. The extirpated pulps were histologically prepared for microscopical evaluation. There was myelin degeneration surrounding the axons and edema in the early posttraumatic stages (17 h). In the later stages (4 to 20 days), the tissues showed varying degrees of inflammation, and neuronal degeneration such as intramyelin edema, aberrant myelin synthesis, and axonal swelling.

Immunophenotypic analysis of human spleen compartments.

Microanatomical compartments of the human spleen are yet under evaluation as most of the present information comes from experiments on animals with different anatomical structures. Immune staining of stromal and blood-born cells by cell surface antigens facilitates the differentiation of functional microanatomical compartmentalization of immune organs, including the spleen. Twenty-two specimens from healthy adult subjects with the average age of 35.6 +/- 13.8 (Range 17 to 58) years were included in this study. Monoclonal antibodies used in this study were supplied from the 5th, 6th and 7th International Workshops and Conferences on Human Leukocyte Differentiation Antigens. Tetraspan antigens presented a rather unique staining pattern in the human spleen, suggesting special roles for each (CD9, CD53, CD63, CD151 and CD231) in certain locations. Sinus lining cells presented a distinctive antigenic profile, sharing both endothelial cell (CD31, CD36, CD54, CD62P, CD102, CD105, CD106 and CD146) and macrophage lineage characteristics. The sheathed capillaries were not restricted to the perifollicular zone alone. Extracellular matrix receptors (CD49 a, CD49 b, CD49 c, CD49 e, CD49f, CD29 and CD44) stained the penicillary arterioles and vascular smooth muscle. These molecules were also found on the vascular endothelium. Leukocyte antigens (CD11a, CD11b, CD22, CD43, CD45, CD45RB, CD45RO and CD50) were mainly expressed in the white and red pulp of the spleen at different intensities, excluding the penicillary arterioles. Activation antigens (CD26, CD71 and CD98) presented a diffuse and broad staining pattern. In conclusion, microanatomical compartmentalization, microcirculation and function of the human spleen were evaluated using a wide panel of monoclonal antibodies.

Vasculogenesis in early human placental villi: an ultrastructural study.

In this study we examined the chorionic villi of 5 normal human placentas at 12-14 weeks of gestation ultrastructurally with regard to differentiation of the vascular components. The aim of the present report is to discuss the factors influencing vasculogenesis (in situ formation of blood vessels) at the ultrastructural level. Our observations have led us to think that the cytotrophoblast influences vasculogenesis in human chorionic villi. Mesenchymal-preendothelial cell groups were always found in very close association with the cytotrophoblast at the periphery of the villi, forming blood vessels. The cytotrophoblast probably attracts mesenchymal cells towards the margin of the villi by secreting vascular endothelial growth factor (VEGF). Once cells attach to the trophoblastic basement membrane they begin to differentiate into endothelial cells. This close structural relation between two cell types (cytotrophoblast and mesenchymal cells) may not be the only mechanism controlling vasculogenesis, but it seems to be one of the factors influencing the differentiation of mesenchymal cells into the endothelial cells of blood vessels in early human chorionic villi.

Ultrastructural findings of bone marrow in a case with malignant osteopetrosis following successful allogeneic bone marrow transplantation.

A nine-month-old female patient suffering from malignant osteopetrosis was evaluated by light and transmission electron microscopic study before and following allogeneic bone marrow transplantation (BMT). Bone marrow specimens were obtained from iliac crest biopsies. Before BMT, the bone marrow had an irregular appearance and was filled with bridging bony trabeculae devoid of cells. Following BMT, the marrow had an almost normal appearance with no myelofibrosis and a relatively regular distribution of hematopoletic cells. The osteocytes were visible in their lacunae in the bone matrix. Presence of bone resorbing and bone forming cell together demonstrated that the bone was beginning to gain its normal dynamic structure. These findings were in accordance with the clinical, laboratory and radiological data which showed the beneficial effect of the therapy.

Serotonergic innervation and serotonin receptor expression of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei.

Pharmacobehavioral studies in experimental animals, and imaging studies in humans, indicate that serotonergic transmission in the amygdala plays a key role in emotional processing, especially for anxiety-related stimuli. The lateral and basolateral amygdaloid nuclei receive a dense serotonergic innervation in all species studied to date. We investigated interrelations between serotonergic afferents and neuropeptide Y (NPY)-producing neurons, which are a subpopulation of inhibitory interneurons in the rat lateral and basolateral nuclei with particularly strong anxiolytic properties. Dual light microscopic immunolabeling showed numerous appositions of serotonergic afferents on NPY-immunoreactive somata. Using electron microscopy, direct membrane appositions and synaptic contacts between serotonin-containing axon terminals and NPY-immunoreactive cellular profiles were unequivocally established. Double in situ hybridization documented that more than 50 %, and about 30-40 % of NPY mRNA-producing neurons, co-expressed inhibitory 5-HT1A and excitatory 5-HT2C mRNA receptor subtype mRNA, respectively, in both nuclei with no gender differences. Triple in situ hybridization showed that individual NPY mRNA-producing interneurons co-express both 5-HT1A and 5-HT2C mRNAs. Co-expression of NPY and 5-HT3 mRNA was not observed. The results demonstrate that serotonergic afferents provide substantial innervation of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei. Studies of serotonin receptor subtype co-expression indicate a differential impact of the serotonergic innervation on this small, but important, population of anxiolytic interneurons, and provide the basis for future studies of the circuitry underlying serotonergic modulation of emotional stimulus processing in the amygdala.

Serotonin transporter knockout and repeated social defeat stress: impact on neuronal morphology and plasticity in limbic brain areas.

Low expression of the human serotonin transporter (5-HTT) gene presumably interacts with stressful life events enhancing susceptibility for affective disorders. 5-Htt knockout (KO) mice display an anxious phenotype, and behavioural differences compared to wild-type (WT) mice are exacerbated after repeated loser experience in a resident-intruder stress paradigm. To assess whether genotype-dependent and stress-induced behavioural differences are reflected in alterations of neuronal morphology in limbic areas, we studied dendritic length and complexity of pyramidal neurons in the anterior cingulate and infralimbic cortices (CG, IL), hippocampus CA1 region, and of pyramidal neurons and interneurons in the lateral (La) and basolateral (BL) amygdaloid nuclei in Golgi-Cox-stained brains of male WT and 5-Htt KO control and loser mice. Spine density was analysed for IL apical and amygdaloid apical and basal pyramidal neuron dendrites. While group differences were absent for parameters analysed in CG, CA1 and amygdaloid interneurons, pyramidal neurons in the IL displayed tendencies to shorter and less spinous distal apical dendrites in 5-Htt KO controls, and to extended proximal dendrites in WT losers compared to WT controls. In contrast, spine density of several dendritic compartments of amygdaloid pyramids was significantly higher in 5-Htt KO mice compared to WT controls. While a tendency to increased spine density was observed in the same dendritic compartments in WT after stress, changes were lacking in stressed compared to control 5-Htt KO mice. Our findings indicate that disturbed 5-HT homeostasis results in alterations of limbic neuronal morphology, especially in higher spinogenesis in amygdaloid pyramidal neurons. Social stress leads to similar but less pronounced changes in the WT, and neuroplasticity upon stress is reduced in 5-Htt KO mice.

Reactive changes in dorsal roots and dorsal root ganglia after C7 dorsal rhizotomy and ventral root avulsion/replantation in rabbits.

Current surgical treatment of spinal root injuries aims at reconnecting ventral roots to the spinal cord while severed dorsal roots are generally left untreated. Reactive changes in dorsal root ganglia (DRGs) and in injured dorsal roots after such complex lesions have not been analysed in detail. We studied dorsal root remnants and lesioned DRGs 6 months after C7 dorsal rhizotomy, ventral root avulsion and immediate ventral root replantation in adult rabbits. Replanted ventral roots were fixed to the spinal cord with fibrin glue only or with glue containing ciliary neurotrophic factor and/or brain-derived neurotrophic factor. Varying degrees of degeneration were observed in the deafferented dorsal spinal cord in all experimental groups. In cases with well-preserved morphology, small myelinated axons extended into central tissue protrusions at the dorsal root entry zone, suggesting sprouting of spinal neuron processes into the central dorsal root remnant. In lesioned DRGs, the density of neurons and myelinated axons was not significantly altered, but a slight decrease in the relative frequency of large neurons and an increase of small myelinated axons was noted (significant for axons). Unexpectedly, differences in the degree of these changes were found between control and neurotrophic factor-treated animals. Central axons of DRG neurons formed dorsal root stumps of considerable length which were attached to fibrous tissue surrounding the replanted ventral root. In cases where gaps were apparent in dorsal root sheaths, a subgroup of dorsal root axons entered this fibrous tissue. Continuity of sensory axons with the spinal cord was never observed. Some axons coursed ventrally in the direction of the spinal nerve. Although the animal model does not fully represent the situation in human plexus injuries, the present findings provide a basis for devising further experimental approaches in the treatment of combined motor/sensory root lesions.