Gravity sedimentation reveals functionally and morphologically different platelets in human blood.

In contrast to red blood cells, platelets float rather than sediment when a column of blood is placed in the gravitational field. By the analogy of erythrocyte sedimentation (ESR), it can be expressed with the platelet antisedimentation rate (PAR), which quantitates the difference in platelet count between the upper and lower halves of the blood column after 1 h of 1 g sedimentation. Venous blood samples from 21 healthy subjects were analyzed for PAR. After a 1-h sedimentation, the upper and lower fractions of blood samples were analyzed for platelet count, mean platelet volume (MPV), immature platelet fraction (IPF), and high-fluorescence IPF (H-IPF). The mechanisms behind platelet flotation were explored by further partitioning of the blood column, time-dependent measurements of platelet count and comparison with ESR. The structure and function of the platelets were assessed by electron microscopy (EM) and atomic force microscopy (AFM), and platelet aggregometry, respectively. Platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation. The area under the curve (AUC) of the whole blood adenosine diphosphate (ADP) aggregation curves showed significant differences between the upper and lower samples (p < .005). AUC in the upper samples of 38% of healthy subjects exceeded the top of the normal range (53-122) suggesting that ascending platelets show an intensified ADP-induced aggregability ex vivo. H-IPF was significantly higher in the upper samples (p < .05). EM and AFM revealed that platelets in the upper samples were larger in volume and contained 1.6 times more alpha granules compared to platelets in the lower samples. Our results indicate that antisedimentation is able to differentiate platelet populations based on their structural and functional properties. Therefore, PAR may be a suitable laboratory parameter in various thromboinflammatory disorders.

It is less known that platelets do not sediment in response to gravitational force but float on the top of the blood column. This phenomenon is called antisedimentation, the rate of which, however, can be different, yet this feature has not been widely studied and used in clinical practice or diagnosis. We tested the idea that antisedimentation of platelets from venous blood samples can be a potential biomarker. We have found that platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation and after 1-h upper and lower fractions develop. Interestingly, the aggregation curves showed significant differences between the upper and lower samples, suggesting that the ascending platelets show ex vivo hyperaggregability. Electron and atomic force microscopy revealed that platelets in the upper samples were larger in volume and contained more alpha granules than platelets in the lower samples. Subsequently, antisedimentation can be used to differentiate platelet populations based on their structural and functional properties; thus, it may be a promising biomarker for various thromboinflammatory disorders.

Etiology-related Degree of Sprouting of Parvalbumin-immunoreactive Axons in the Human Dentate Gyrus in Temporal Lobe Epilepsy.

In the present study, we examined parvalbumin-immunoreactive cells and axons in the dentate gyrus of surgically resected tissues of therapy-resistant temporal lobe epilepsy (TLE) patients with different etiologies. Based on MRI results, five groups of patients were formed: (1) hippocampal sclerosis (HS), (2) malformation of cortical development, (3) malformation of cortical development + HS, (4) tumor-induced TLE, (5) patients with negative MRI result. Four control samples were also included in the study. Parvalbumin-immunoreactive cells were observed mostly in subgranular location in the dentate hilus in controls, in tumor-induced TLE, in malformation of cortical development and in MR-negative cases. In patients with HS, significant decrease in the number of hilar parvalbumin-immunoreactive cells and large numbers of ectopic parvalbumin-containing neurons were detected in the dentate gyrus' molecular layer. The ratio of ectopic/normally-located cells was significantly higher in HS than in other TLE groups. In patients with HS, robust sprouting of parvalbumin-immunoreactive axons were frequently visible in the molecular layer. The extent of sprouting was significantly higher in TLE patients with HS than in other groups. Strong sprouting of parvalbumin-immunoreactive axons were frequently observed in patients who had childhood febrile seizure. Significant correlation was found between the level of sprouting of axons and the ratio of ectopic/normally-located parvalbumin-containing cells. Electron microscopy demonstrated that sprouted parvalbumin-immunoreactive axons terminate on proximal and distal dendritic shafts as well as on dendritic spines of granule cells. Our results indicate alteration of target profile of parvalbumin-immunoreactive neurons in HS that contributes to the known synaptic remodeling in TLE.

Surgical stress and cytoskeletal changes in lens epithelial cells following manual and femtosecond laser-assisted capsulotomy.

AIM: To study the effect of mechanical stress on the cytoskeleton in lens epithelial cells following conventional phacoemulsification surgery (CPS) and femtosecond laser-assisted cataract surgery (FLACS). METHODS: The cytoskeleton of the epithelial cells of the anterior lens capsules (ALC) removed by CPS and FLACS was examined by immunohistochemistry. Expression of the intermediate filament, glial fibrillary acidic protein (GFAP), and glutamine synthetase (GS) immunoreactivity were detected. In order to map the actin network of cells, fluorescently labeled phalloidin was used. The samples were examined using confocal laser scanning microscopy. RESULTS: GFAP expression was visible in a larger number of the epithelial cells after CPS compared to FLACS. In CPS sample's epithelial cells, GFAP immunoreactivity indicated robust morphological change. Regarding the actin filaments, the presence of tubular elements connecting epithelial cells, regular actin pattern and marked cortical network after CPS were found. Following FLACS, the actin cytoskeleton of the epithelial cells remained densely structured, and the tubular elements were undetectable, however, the above-mentioned regular actin pattern and the marked cortical network were visible. CONCLUSION: The conventional removal of the ALC induces more robust changes of the cytoskeleton of the lens epithelial cells.

[Postoperative outcome of surgical interventions for epilepsy between 2005 and 2016 at the Epilepsy Center of Pécs]. / Epilepsziasebészeti beavatkozások eredményei a Pécsi Epilepszia Centrumban 2005 és 2016 között.

INTRODUCTION: Epilepsy as a chronic, severe neurologic disease significantly influences the quality of life of the epileptic patients. In candidates well selected for surgery, the seizure freedom is realistically achievable, and the quality of life can be further improved with complex individual rehabilitation. AIM: We aimed to evaluate the postoperative outcome of patients who underwent epilepsy surgery between 2005 and 2016 at the Epilepsy Center at Pécs. METHOD: We evaluated seizure status at regular follow-up visits after surgery and the quality of life using questionnaires focusing on employment and social status. RESULTS: 76% of the 72 patients who underwent surgical resection for epilepsy were free from disabling seizures , and 10% had rare disabling seizures (almost seizure-free), 7% experienced worthwhile improvement and 7% had no worthwhile improvement. Comparing the employment status of patients free from disabling seizures to patients not free from disabling seizures, we found that the employment status is significantly influenced by seizure freedom (p<0.01, Fisher's exact test). While 67% of seizure-free patients were employed, only 19% of patients not free from disabling seizures were hired. CONCLUSION: Our results resemble the international tendencies and success rate, proving epilepsy surgery as an available, valid and effective treatment in well selected patients. Orv Hetil. 2019; 160(7): 270-278.

Cell death and survival following manual and femtosecond laser-assisted capsulotomy in age-related cataract.

AIM: To study molecular and morphological changes in lens epithelial cells following femtosecond laser-assisted and manually performed continuous curvilinear capsulotomy (CCC) in order to get information about these methods regarding their potential role in the induction of development of secondary cataract. METHODS: Anterior lens capsules (ALC) were removed from 40 patients with age-related cataract by manual CCC and by femtosecond laser-assisted capsulotomy (FLAC). Samples removed by manual CCC were assorted in group 1, FLAC samples were classified in group 2. Morphology of lens epithelial cells was examined with light and electron microscopes. Following capsulotomy, expressions of p53, Bcl-2 and cyclin D1 genes were analyzed with reverse transcriptase polymerase chain reaction. Immunohistochemistry was used to detect the pro-apoptotic p53 in the epithelial cells. RESULTS: Light and electron microscopic examination showed that ALC of group 1 contained more degenerating cells following manual CCC than after FLAC. The expression level of p53 was higher after manual than laser-assisted surgery. Immunocytochemistry indicated significantly higher number of cells containing p53 protein in the manual CCC group than following FLAC. Bcl-2 and cyclin D1 gene expression levels were slightly lower following manual CCC than after FLAC, but the difference was not significant. CONCLUSION: Manually removed ALC shows slightly, but not significantly larger damage due to the mechanical stretching and pulling of the capsule than those removed using FLAC.

Antimicrobial and Virulence-Modulating Effects of Clove Essential Oil on the Foodborne Pathogen Campylobacter jejuni.

Our study investigated the antimicrobial action of clove (Syzygium aromaticum) essential oil (EO) on the zoonotic pathogen Campylobacter jejuni After confirming the clove essential oil's general antibacterial effect, we analyzed the reference strain Campylobacter jejuni NCTC 11168. Phenotypic, proteomic, and transcriptomic methods were used to reveal changes in cell morphology and functions when exposed to sublethal concentrations of clove EO. The normally curved cells showed markedly straightened and shrunken morphology on the scanning electron micrographs as a result of stress. Although, oxidative stress, as a generally accepted response to essential oils, was also present, the dominance of a general stress response was demonstrated by reverse transcription-PCR (RT-PCR). The results of RT-PCR and two-dimensional (2D) PAGE revealed that clove oil perturbs the expression of virulence-associated genes taking part in the synthesis of flagella, PEB1, PEB4, lipopolysaccharide (LPS), and serine protease. Loss of motility was also detected by a phenotypic test. Bioautographic analysis revealed that besides its major component, eugenol, at least four other spots of clove EO possessed bactericidal activity against C. jejuni Our findings show that clove EO has a marked antibacterial and potential virulence-modulating effect on C. jejuni IMPORTANCE: This study demonstrates that the components of clove essential oil influence not only the expression of general stress genes but also the expression of virulence-associated genes. Based on this finding, alternative strategies can be worked on to control this important foodborne pathogen.

Characterization of neurons in the cortical white matter in human temporal lobe epilepsy.

The aim of the present work was to characterize neurons in the archi- and neocortical white matter, and to investigate their distribution in mesial temporal sclerosis. Immunohistochemistry and quantification of neurons were performed on surgically resected tissue sections of patients with therapy-resistant temporal lobe epilepsy. Temporal lobe tissues of patients with tumor but without epilepsy and that from autopsy were used as controls. Neurons were identified with immunohistochemistry using antibodies against NeuN, calcium-binding proteins, transcription factor Tbr1 and neurofilaments. We found significantly higher density of neurons in the archi- and neocortical white matter of patients with temporal lobe epilepsy than in that of controls. Based on their morphology and neurochemical content, both excitatory and inhibitory cells were present among these neurons. A subset of neurons in the white matter was Tbr-1-immunoreactive and these neurons coexpressed NeuN and neurofilament marker SMI311R. No colocalization of Tbr1 was observed with the inhibitory neuronal markers, calcium-binding proteins. We suggest that a large population of white matter neurons comprises remnants of the subplate. Furthermore, we propose that a subset of white matter neurons was arrested during migration, highlighting the role of cortical maldevelopment in epilepsy associated with mesial temporal sclerosis.

Correlation between calbindin expression in granule cells of the resected hippocampal dentate gyrus and verbal memory in temporal lobe epilepsy.

Calbindin expression of granule cells of the dentate gyrus is decreased in temporal lobe epilepsy (TLE) regardless of its etiology. In this study, we examined the relation between reduction of calbindin immunoreactivity and the verbal and visuo-spatial memory function of patients with TLE of different etiologies. Significant linear correlation was shown between calbindin expression and short-term and long-term percent retention and retroactive interference in auditory verbal learning test (AVLT) of patients including those with hippocampal sclerosis. In addition, we found significant linear regression between calbindin expression and short-term and long-term percent retention of AVLT in patients whose epilepsy was caused by malformation of cortical development or tumor and when no hippocampal sclerosis and substantial neuronal loss were detected. Together with the role of calbindin in memory established in previous studies on calbindin knock-out mice, our results suggest that reduction of calbindin expression may contribute to memory impairments of patients with TLE, particularly, when neuronal loss is not significant.

Role of endogenous pituitary adenylate cyclase activating polypeptide (PACAP) in myelination of the rodent brain: lessons from PACAP-deficient mice.

Pituitary adenylate-cyclase activator polypeptide (PACAP), as a consequence of its effect on the elevation of intracellular cAMP level, strongly influences brain development including myelination. While proliferation of oligodendroglial progenitors is stimulated by PACAP applied in vitro, their differentiation is inhibited. However, the in vivo role of PACAP on myelination has never been examined. In the present study the role of endogenous PACAP in myelination was examined in PACAP-deficient mice, in several areas of the brain with a special attention to the cerebral cortex. In young postnatal and adult mice myelination was studied with immunohistochemistry detecting a protein present in the myelin sheath, the myelin basic protein, with Luxol Fast Blue staining and with electron microscopy. Results obtained in PACAP-deficient mice were compared to age-matched wild type controls. We found that the sequence of myelination in the PACAP-deficient animals was similar to that observed in controls. According to this, in both PACAP-deficient and wild type mice, the somatosensory cortex was myelinated before motor areas that preceded the myelination of associational cortical areas. Archicortical associational areas such as the cingulate cortex were myelinated before neocortical areas. Myelination in the corpus callosum followed the known rostro-caudal direction in both PACAP-deficient and wild type animals, and the ventrolateral part of the corpus callosum was myelinated earlier than the dorsomedial part in both groups. In contrast to the similarity in its sequence, striking difference was found in the onset of myelination that started earlier in PACAP-deficient mice than in wild type controls in all of the examined brain regions, including cerebral archi- and neocortex. The first myelinated axons in each of the examined brain regions were observed earlier in the PACAP-deficient mice than in controls. When age-matched animals of the two groups were compared, density of myelinated fibers in the PACAP-deficient mice was higher than in controls in all of the examined areas. We propose that endogenous PACAP exerts an inhibitory role on myelination in vivo. Since myelin sheath of the central nervous system contains several factors blocking neurite outgrowth, inhibition of myelination by PACAP gives time for axonal development and synapse formation, and therefore, strengthens neuronal plasticity.

Degree and pattern of calbindin immunoreactivity in granule cells of the dentate gyrus differ in mesial temporal sclerosis, cortical malformation- and tumor-related epilepsies.

A loss of calbindin immunoreactivity in granule cells of the hippocampal dentate gyrus is a characteristic feature of temporal lobe epilepsy with hippocampal sclerosis. Whether decreased calbindin expression is unique to the hippocampal sclerosis associated with cryptogenic temporal lobe epilepsy, or also occurs in tumor- or malformation-related epilepsy, is unknown. We show that calbindin immunoreactivity in granule cells has been decreased in epilepsy regardless of its etiology. In cases of cortical malformations or hippocampal sclerosis, calbindin immunoreactivity was undetectable in most granule cells. In tumor-related resections, in patients who had a long history of epileptic seizures, calbindin was detected only in one-third of granule cells. Regardless of etiology, calbindin expression correlated with age of onset and with duration of the epilepsy. In contrast to tumor-induced epilepsy, where calbindin-immunoreactive granule cells were equally distributed in the granule cell layer, in hippocampal sclerosis and malformation-related epilepsy, two-thirds of calbindin-immunoreactive granule cells were located in the outer half and only one-third in the inner half of the layer. Developmentally, granule cells at the border of the molecular layer are ontogenetically the oldest, and those at the border of the hilus are the youngest. The reduction of calbindin immunoreactivity in ontogenetically younger granule cells highlights the deleterious effect of early occurring epilepsy and initial early precipitating injury, including febrile seizures that may substantially affect developing immature granule cells, but less the earlier born matured ones.

Survival of mossy cells of the hippocampal dentate gyrus in humans with mesial temporal lobe epilepsy.

OBJECT: Hippocampal sclerosis can be identified in most patients with mesial temporal lobe epilepsy (TLE). Surgical removal of the sclerotic hippocampus is widely performed to treat patients with drug-resistant mesial TLE. In general, both epilepsy-prone and epilepsy-resistant neurons are believed to be in the hippocampal formation. The hilar mossy cells of the hippocampal dentate gyrus are usually considered one of the most vulnerable types of neurons. The aim of this study was to clarify the fate of mossy cells in the hippocampus in epileptic humans. METHODS: Of the 19 patients included in this study, 15 underwent temporal lobe resection because of drug-resistant TLE. Four patients were used as controls because they harbored tumors that had not invaded the hippocampus and they had experienced no seizures. Histological evaluation of resected hippocampal tissues was performed using immunohistochemistry. RESULTS: Mossy cells were identified in the control as well as the epileptic hippocampi by using cocaine- and amphetamine-regulated transcript peptide immunohistochemistry. In most cases the number of mossy cells was reduced and thorny excrescences were smaller in the epileptic hippocampi than in controls; however, there was a significant loss of pyramidal cells and a partial loss of granule cells in the same epileptic hippocampi in which mossy cell loss was apparent. The loss of mossy cells could be correlated with the extent of hippocampal sclerosis, patient age at seizure onset, duration of epilepsy, and frequency of seizures. CONCLUSIONS: In many cases large numbers of mossy cells were present in the hilus of the dentate gyrus when most pyramidal neurons of the CA1 and CA3 areas of the Ammon's horn were lost, suggesting that mossy cells may not be more vulnerable to epileptic seizures than the hippocampal pyramidal neurons.

Fetal development of membrane water channel proteins aquaporin-1 and aquaporin-4 in the human brain.

Aquaporin-1 and aquaporin-4, water channel membrane proteins reported in both experimental animals and in adult humans, have been detected in different, non-overlapping areas of the central nervous system. This immunohistochemical study describes the developmental expression pattern of the water channel membrane proteins, aquaporin-1 and aquaporin-4, in various structures of human fetal brain over the gestational period of 14-40 weeks. Aquaporin-1 immunostaining was exclusively found in the epithelial cells of the choroid plexus from the 14th gestational week, and the staining pattern altered slightly over time. At week 14, immunostaining appeared only in the apical cell membranes. By the 18th gestational week, the entire plasma membrane of these apical cells was immunopositive, as well as was the cytosol. These changes in immunoreactivity indicate an increasing production of aquaporin-1 in the epithelial cells during the period between the 14th and 24th weeks of gestation. Aquaporin-4 immunostaining was first detected in the archicortex, from gestational week 14 and was detected in the neocortex, 6-7 weeks later. Immunostained structures were always astrocytes, particularly the astrocytic endfeet in the ventricular wall, at the developing ependymal lining, at the pial surface, and around the capillaries. Neuronal labeling was not observed. These results in human fetal brain lend morphological support to the previous findings that aquaporin-1 and aquaporin-4 play different roles in the regulation of the water homeostasis of the brain.

p73 and Reelin in Cajal-Retzius cells of the developing human hippocampal formation.

In the fetal human hippocampus, Cajal-Retzius (CR) cells coexpress p73, a p53-family member involved in cell survival and apoptosis, and the glycoprotein reelin, crucial for radial migration. We distinguish two populations of putative CR cells. (1). p73/reelin expressing cells appear around 10 gestational weeks (GW) at the cortico-choroid border in the temporal horn of the lateral ventricle (the ventral cortical hem) and occupy the marginal zone (MZ) overlying the ammonic and dentate primordia. (2). Additional p73-positive cells appear from 14 GW onward in the neuroepithelium near the dentate-fimbrial boundary and spread toward the pial surface, flanking the migrating secondary dentate matrix. From 13 to 17 GW, large parts of the dentate gyrus are almost devoid of CR cells. p73/Reelin-positive CR cells appear in the MZ of the suprapyramidal blade at 16 GW and around 21 GW in the infrapyramidal blade. The p73-positive cells of the dentate-fimbrial boundary express reelin when they are close to the pial surface, suggesting that they differentiate into CR cells of the infrapyramidal blade. Reelin-positive, p73-negative interneurons are prominent in the prospective strata lacunosum-moleculare and radiatum of cornu ammonis as early as 14 GW; in the dentate molecular layer and hilus they appear around midgestation. We propose that CR cells of the human hippocampal formation belong to two distinct cell populations: an early one derived from the ventral cortical hem and mainly related to migration of the ammonic and dentate plates and a later appearing one derived from the dentate-fimbrial neuroepithelium, which may be related to the protracted neurogenesis and migration of dentate granule cells, particularly of the infrapyramidal blade.

Reelin-expressing neurons in the postnatal and adult human hippocampal formation.

Reelin plays a major role in the development of laminated brain structures. In the developing neocortex and hippocampus, Reelin is secreted by Cajal-Retzius cells in the marginal zone. In the present report, we characterize Reelin-immunoreactive neurons in the perinatal and adult human hippocampal formation. Two main populations of cells are described: Cajal-Retzius cells and interneurons. Cajal-Retzius cells are defined as neurons that coexpress Reelin and p73, a nuclear protein of the p53 family. Colocalization experiments of p73 with calcium-binding proteins indicate that most Cajal-Retzius cells express calretinin, but not calbindin. Cajal-Retzius cell density decreases dramatically during the postnatal period, although a few Reelin/p73-positive neurons are still found in the adult. At birth, Reelin-positive, p73-negative neurons are present in all layers of the hippocampal formation. Their morphology and localization indicate that they belong to a heterogeneous population of interneurons. They are numerous in the strata lacunosum-moleculare and radiatum of CA1-CA3, in the hilus, and in the molecular layer of the dentate gyrus, but less common in stratum oriens and alveus, and rare in the principal cell layers. Subpopulations of Reelin-positive interneurons express calretinin or calbindin. The packing density of Reelin-positive cells decreases postnatally, which may be related to the disappearance of Cajal-Retzius cells and to the growth of the hippocampal formation. The presence of Reelin-immunoreactive cells in the adult hippocampal formation indicates that Reelin is not restricted to development but that it may have additional functions in adult life.

Expression of p73 and Reelin in the developing human cortex.

Cajal-Retzius (CR) cells of the developing neocortex secrete Reelin (Reln), a glycoprotein involved in neuronal migration. CR cells selectively express p73, a p53 family member implicated in cell survival and apoptosis. Immunocytochemistry in prenatal human telencephalon reveals a complex sequence of migration waves of p73- and Reln-immunoreactive (IR) neurons into the cortical marginal zone (MZ). At early preplate stages, p73/Reln-IR cells arise in distinct sectors of the telencephalon, including cortical primordium and ganglionic eminences. After the appearance of the cortical plate, further p73/Reln-IR cells originate in the medial periolfactory forebrain. In addition, p73 marks a novel cell population that appears at the choroid-cortical junction or cortical hem before the emergence of the dorsal hippocampus. A pronounced mediolateral gradient in the density of p73/Reln-IR neurons in the neocortical MZ at 8 gestational weeks suggests that a subset of CR cells migrate tangentially from cortical hem and taenia tecta into neocortical territory. This hypothesis is supported by the absence of p73-transcripts in prospective neocortex of p73-/-mice at embryonic day 12 (E12), whereas they are present in cortical hem and taenia tecta. In the p73-/- preplate, Reln is faintly expressed in a calretinin-positive cell population, not present in this form in the E12 wild-type cortex. At P2, Reln-IR CR cells are undetectable in the p73-/- cortex, whereas Reln-expression in interneurons is unchanged. Our results point to a close association between p73 and Reln in CR cells of the developing neocortex, with a partial dissociation in early preplate and basal telencephalon, and to a p73-mediated role of the cortical hem in neocortical development.

Filament size influences temperature changes and brain damage following middle cerebral artery occlusion in rats.

Postischemic spontaneous hyperthermia as a complication of occlusion of the middle cerebral artery with an intraluminal filament has been observed by some authors, but many other reports do not discuss this factor. The possible reasons why some of the authors have not seen severe hyperthermia in their experiments include differences in surgical technique, the strain of animals, the type of the anesthesia, and the occluder filament. The aim of this study was to examine the changes in the core temperature of rats using different types of filaments. The middle cerebral artery was occluded for 2 h with three different types of filaments. The changes in the temperature were continuously monitored during occlusion and for the next 4 h. Groups with uncontrolled hyperthermia and with controlled normal core temperature were used. In addition, the necrotic and penumbral areas were measured 4 and 48 h after the ischemia in both groups. Spontaneous postischemic hyperthermia was detected using all types of filaments. A close correlation was found between the size of the occluder filament and the time-course and degree of hyperthermia. Moreover, the size of the filament correlated well with the size of the infarct at both 4 and 48 h after the occlusion. We suggest that filament size is a major contributor to the degree of hyperthermia and the development of brain damage in the middle cerebral artery occlusion model. Our results call attention to the need to standardize the methods used to screen for therapeutic agents for stroke.