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From the beginning of recorded history through the present day, dermatologic disorders have been treated with ethnomedicine remedies. We present the ethnodermatologic practices in Transylvania, Romania. We conducted ethnomedicine surveys in 35 villages in Transylvania (2007-2019). The 650 people interviewed were questioned about the treatment of dermatologic disorders by drugs derived from plant, animal, human, or other origins. Collected data were compared to earlier records of the regions and other European countries, completed with relevant pharmacologic studies of some plants. A total of 180 drugs were documented for 45 skin problems, including 112 plants, 1 fungus, 19 animals, 5 humans, and 43 other materials used in 11 preparation forms. Among these, 144 drugs were mentioned in humans, 10 in veterinary medicines, and 26 included in both therapies with overlapping human/animal (eg, Petroselinum crispum) and specific uses (eg, Daphne mezereum, Scrophularia nodosa). Compared to data from other countries, the local use of 32 plants and various animals and minerals was described only in the study area. The present study demonstrates that ethnomedicine practices are a valuable source of knowledge for skin diseases and highlight the relevance of fieldwork in the selected regions of Transylvania.

PACAP has well-known neuroprotective potential including traumatic brain injury (TBI). Its level is up-regulated following various insults of the CNS in animal models. A few studies have documented alterations of PACAP levels in human serum. The time course of post-ictal PACAP levels, for example, show correlation with migraine severity. Very little is known about the course of PACAP levels following CNS injury in humans and the presence of PACAP has not yet been detected in cerebrospinal fluid (CSF) of subjects with severe TBI (sTBI). The aim of the present study was to determine whether PACAP occurs in the CSF and plasma (Pl) of patients that suffered sTBI and to establish a time course of PACAP levels in the CSF and Pl. Thirty eight subjects with sTBI were enrolled with a Glasgow Coma Scale ≤8 on admission. Samples were taken daily, until the time of death or for maximum 10 days. Our results demonstrated that PACAP was detectable in the CSF, with higher concentrations in patients with TBI. PACAP concentrations markedly increased in both Pl and CSF in the majority of patients 24-48h after the injury stayed high thereafter. In cases of surviving patients, Pl and CSF levels displayed parallel patterns, which may imply the damage of the blood-brain barrier. However, in patients, who died within the first week, Pl levels were markedly higher than CSF levels, possibly indicating the prognostic value of high Pl PACAP levels.

Advanced magnetic resonance imaging (MRI) methods were shown to be able to detect the subtle structural consequences of mild traumatic brain injury (mTBI). The objective of this study was to investigate the acute structural alterations and recovery after mTBI, using diffusion tensor imaging (DTI) to reveal axonal pathology, volumetric analysis, and susceptibility weighted imaging (SWI) to detect microhemorrhage. Fourteen patients with mTBI who had computed tomography with negative results underwent MRI within 3 days and 1 month after injury. High resolution T1-weighted imaging, DTI, and SWI, were performed at both time points. A control group of 14 matched volunteers were also examined following the same imaging protocol and time interval. Tract-Based Spatial Statistics (TBSS) were performed on DTI data to reveal group differences. T1-weighted images were fed into Freesurfer volumetric analysis. TBSS showed fractional anisotropy (FA) to be significantly (corrected p

To examine the changes in MR parameters derived from diffusion weighted imaging (DWI) biexponential analysis in an in vivo intracellular brain oedema model, and to apply electron microscopy (EM) to shed more light on the morphological background of MR-related observations.Intracellular oedema was induced in ten male Wistar rats (380-450g) by way of water load, using a 20% body weight intraperitoneal injection of 140mmol/L dextrose solution. A 3T MRI instrument was used to perform serial DWI, and MR specroscopy (water signal) measurements. Following the MR examination the brains of the animals were analyzed for EM.Following the water load induction, apparent diffusion coefficient (ADC) values started declining from 724±43μm(2)/s to 682±26μm(2)/s (p0.0001). ADC-fast values dropped from 948±122 to 840±66μm(2)/s (p0.001). ADC-slow showed a decrease from 226±66 to 191±74μm(2)/s (p0.05). There was a shift from the slow to the fast component at 110min time point. The percentage of the fast component demonstrated moderate, yet significant increase from 76.56±7.79% to 81.2±7.47% (p0.05). The water signal was increasing by 4.98±3.52% compared to the base line (p0.01). The results of the E.M. revealed that water was detected intracellularly, within astrocytic preivascular end-feet and cell bodies.The unexpected volume fraction changes (i.e. increase in fast component) detected in hypotonic oedema appear to be substantially different from those observed in stroke. It may suggest that ADC decrease in stroke, in contrast to general presumptions, cannot be explained only by water shift from extra to intracellular space (i.e. intracellular oedema).

Pituitary adenylate cyclase activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. In addition to its more classic role as a neuromodulator, PACAP functions as a neurotrophic factor. Several neurotrophic factors have been shown to play an important role in the endogenous response following both cerebral ischemia and traumatic brain injury and to be effective when given exogenously. A number of studies have shown the neuroprotective effect of PACAP in different models of ischemia, neurodegenerative diseases and retinal degeneration. The aim of this review is to summarize the findings on the neuroprotective potential of PACAP in models of different traumatic nerve injuries. Expression of endogenous PACAP and its specific PAC1 receptor is elevated in different parts of the central and peripheral nervous system after traumatic injuries. Some experiments demonstrate the protective effect of exogenous PACAP treatment in different traumatic brain injury models, in facial nerve and optic nerve trauma. The upregulation of endogenous PACAP and its receptors and the protective effect of exogenous PACAP after different central and peripheral nerve injuries show the important function of PACAP in neuronal regeneration indicating that PACAP may also be a promising therapeutic agent in injuries of the nervous system.

The present study was undertaken to reveal the influence of intracerebroventricular (ICV) benzamil on the dynamics of brain water accumulation in hyponatremic rats. Parameters of brain water homeostasis were continuously monitored, using in vivo magnetic resonance imaging (MRI) methods. The results were compared with those obtained in a previous study by tissue desiccation. A 3-T MRI instrument was applied to perform serial diffusion-weighted imaging to measure the apparent diffusion coefficient (ADC) and MR spectroscopy to determine water signal. A decrease of ADC is thought to represent an increase of intracellular water, whereas water signal is used to quantify brain water content. Five groups of male Wistar rats were studied as follows: normonatremic, native animals (group NN, n = 7), hyponatremic animals (group HN, n = 8), hyponatremic animals treated with ICV benzamil (group HNB, n = 8), hyponatremic animals treated with ICV saline (group HNS, n = 5) and normonatremic animals treated with ICV benzamil (group NNB, n = 5). Hyponatremia was induced by intraperitoneal administration of 140 mmol/l dextrose solution in a dose of 20% of body weight. Benzamil hydrochloride (4 μg) was injected ICV to the treated animals. During the course of hyponatemia, ADC declined steadily from the baseline (100%) to reach a minimum of 92.32 ± 3.20% at 90 min (p

We examined the neuro/axono-protective potential of a novel poly (ADP-ribose) polymerase (PARP) inhibitor L-2286 in a rat impact acceleration brain injury model. Male Wistar rats (n = 70) weighing 300-350 grams were used to determine the most effective intracerebroventricular (i.c.v.) dose of L-2286 administered 30 min after injury, and to test the neuroprotective effect at two time points (immediately, and 30 min after injury). The neuroprotective effect of L-2286 was tested using immunohistochemical (amyloid precursor protein and mid-sized mouse anti-neurofilament clone RMO-14.9 antibody) and behavioral tests (beam-balance, open-field and elevated plus maze). At both time-points, a 100 microg/rat dose of i.c.v. L-2286 significantly (p < 0.05) reduced the density of damaged axons in the corticospinal tract and medial longitudinal fascicle compared to controls. In the behavioral tests, treatment 30 min post-injury improved motor function, while the level of anxiety was reduced in both treatment protocols.

Calcium-induced, calpain-mediated proteolysis (CMSP) has recently been implicated to the pathogenesis of diffuse (traumatic) axonal injury (TAI). Some studies suggested that subaxolemmal CMSP may contribute to axolemmal permeability (AP) alterations observed in TAI. Seeking direct evidence for this premise we investigated whether subaxolemmal CMSP may contribute to axolemmal permeability alterations (APA) and pre-injury calpain-inhibition could reduce AP in a rat model of TAI. Horseradish peroxidase (HRP, a tracer that accumulates in axons with APA) was administered one hour prior to injury into the lateral ventricle; 30 min preinjury a single tail vein bolus injection of 30 mg/kg MDL-28170 (a calpain inhibitor) or its vehicle was applied in Wistar rats exposed to impact acceleration brain injury. Histological detection of traumatically injured axonal segments accumulating HRP and statistical analysis revealed that pre-injury administration of the calpain inhibitor MDL-28170 significantly reduced the average length of HRP-labeled axonal segments. The axono-protective effect of pre-injury calpain inhibition recently demonstrated with classical immunohistochemical markers of TAI was further corroborated in this experiment; significant reduction of the length of labeled axons in the drug-treated rats implicate CMSP in the progression of altered AP in TAI.

This review summarizes protein biomarkers in mild and severe traumatic brain injury in adults and children and presents a strategy for conducting rationally designed clinical studies on biomarkers in head trauma.We performed an electronic search of the National Library of Medicine's MEDLINE and Biomedical Library of University of Pennsylvania database in March 2008 using a search heading of traumatic head injury and protein biomarkers. The search was focused especially on protein degradation products (spectrin breakdown product, c-tau, amyloid-beta(1-42)) in the last 10 years, but recent data on "classical" markers (S-100B, neuron-specific enolase, etc.) were also examined.We identified 85 articles focusing on clinical use of biomarkers; 58 articles were prospective cohort studies with injury and/or outcome assessment.We conclude that only S-100B in severe traumatic brain injury has consistently demonstrated the ability to predict injury and outcome in adults. The number of studies with protein degradation products is insufficient especially in the pediatric care. Cohort studies with well-defined end points and further neuroproteomic search for biomarkers in mild injury should be triggered. After critically reviewing the study designs, we found that large homogenous patient populations, consistent injury, and outcome measures prospectively determined cutoff values, and a combined use of different predictors should be considered in future studies.

Although it is well known that traumatic brain injury (TBI) evokes traumatic axonal injury (TAI) within the brain, TBI-induced axonal damage in the spinal cord (SC) has been less extensively investigated. Detection of such axonal injury in the spinal cord would further the complexity of TBI while also challenging some functional neurobehavioral endpoints frequently used to assess recovery in various models of TBI. To assess TAI in the spinal cord associated with TBI, we analyzed the craniocervical junction (CCJ), cervico-thoracic (CT), and thoraco-lumber (ThL) spinal cord in a rodent model of impact acceleration of TBI of varying severities. Rats were transcardially fixed with aldehydes at 2, 6, and 24 h post-injury (n = 36); each group included on sham-injured rodent. Semi-serial vibratome sections were reacted with antibodies targeting TAI via alteration in cytoskeletal integrity or impaired axonal transport. Consistent with previous observations in this model, the CCJ contained numerous injured axons. Immunoreactive, damaged axonal profiles were also detected as caudal, as the ThL spinal cord displayed morphological characteristics entirely consistent with those described in the brainstem and the CCJ. Quantitative analyses demonstrated that the occurrence and extent of TAI is positively associated with the impact/energy of injury and negatively with the distance from the brainstem. These observations show that TBI can evoke TAI in regions remote from the injury site, including the spinal cord itself. This finding is relevant to shaken baby syndrome as well as during the analysis of data in functional recovery in various models of TBI.

Background information. For explanation of the formation of ‘dark’ neurons, an enigmatic phenomenon in neuropathology, we hypothesized recently that all spaces between the ultrastructural elements visible in the traditional transmission electron microscope are filled with a gel structure that stores free energy in the form of non-covalent interactions, is continuous in the whole soma—dendrite domains of neurons, and is capable of whole-cell phase transition. This hypothesis was deduced from the fact that ‘dark’ neurons can be formed, even under conditions extremely unfavourable for enzyme-mediated biochemical processes, if initiated by a physical damage. In order to gain further information on this gel structure, we perfused transcardially rats for 5 min with physiological saline containing 1 mM SDS before the perfusion of a fixative for electron microscopy. Results. Dramatic compaction of visibly intact ultrastructural elements was caused in the whole soma—dendrite domains of thinly scattered neurons (‘dark’ neurons), whereas substantial cytoplasmic swelling and patchy ultrastructural disintegration occurred in numerous other neurons (‘light’ neurons). Similar morphological changes were observed in scattered astrocytes, oligodendrocytes, pericytes and endothelial cells. Conclusions. These observations: (i) support the existence of the above intracellular gel structure in neurons; (ii) allow the conclusion that this gel structure is present in the form of an ubiquitous trabecular network surrounded by a confluent system of fluid cytoplasm; (iii) draw attention to the possibility that the previous two statements also apply to other cell types of the brain tissue; and (iv) suggest that pressure-induced direct channels exist between neurons and astrocytes.

The sugar composition of the floral nectar was studied in 22 mainly Hungarian local quince cultivars in 2004. The material for investigation was taken from the quince genebank of the Research and Extension Centre for Fruitgrowing, Ujfeherto, Hungary. The three main sugar components of the nectar were glucose, fructose and sucrose. Sucrose was present in the greatest amount in the nectar of all investigated quince cultivars. Glucose which is the least sweet among the three main nectar sugars for bees was present in the least amount. The nectar of most cultivars contained more fructose than glucose. The amount of the total nectar sugars was the lowest in cv. 'Pear-shaped Noszvaji' and the highest in cv. 'Kuti'. According to the Baker-quotient (S/F+G), the majority of the quince cultivars belonged to the sucrose dominant category. However, the cultivars 'Mezőkovesdi', 'Apple-shaped Dunabogdany' and 'Vali' had sucrose-rich nectar. The concentration of floral secretory product in all cultivars reached the threshold of bee visitation. The nectar refraction was lower at the end of bloom than in full bloom while flowers contained more nectar at the previous stage. The above results are important from the viewpoint of pollination biology, apiculture and defence against fire blight.

The knowledge of nectar composition in entomogamous fruits, including sour cherry cultivars, is of high importance, since pollinator insects have their own taste-preference, which can influence pollination, and hence fruit yield. The floral secretory product of several sour cherry cultivars was studied from 1997 to 2000, in order to determine the floral insect attraction of the cultivars. Here the nectar composition of two major cultivars, an autofertile ('Ujfehertoi furtos') and an autosterile one ('Pandy 48'), will be presented. Samples were collected at the Research and Extension Centre for Fruitgrowing, Ujfeherto, Hungary. Nectar was drained from the flowers with a microcapillary, following a 24-hour isolation. Sugar components of the floral secretory product were determined by thin layer chromatography, quantitative analysis was carried out by densitometry. When evaluating the results, air temperatures and precipitation data during sample taking were also taken into consideration. The nectar of both studied cultivars contains all three major sugar components: sucrose, glucose and fructose. In most seasons the total sugar concentration reached the apicultural threshold value (100 mg/ml). On the basis of the sucrose/(glucose+fructose) quotient the nectar of 'Ujfehertoi furtos' belonged to the sucrose-rich group each year, like the majority of sour cherry cultivars, whereas the secretory product of 'Pandy 48' could be classified into the sucrose-dominant category in one of the seasons. The nectar sugar composition of 'Ujfehertoi furtos' varied to a great extent according to the seasons, while the sugar components in the nectar of 'Pandy 48' were rather stable in the four seasons of study, their amounts changing to a small extent, effected by microclimatic factors.

The sugar composition of floral nectar was studied in 17 sour cherry cultivars in Ujfeherto in 1997-2000. All samples contained the three most frequent sugar components: glucose, fructose and sucrose, similarly to our previous data. The ranking of the floral nectar in sour cherry cultivars based on sucrose content was the following: 'Ujfehertoi furtos' and 'Pandy 48' reached the threshold value of bee visitation in at least three seasons, and a significant amount of sucrose was detected in the flowers of 'Erdi jubileum AB' and 'Erdi bőtermő' as well. The above cultivars proved to be the most valuable in Ujfeherto from the viewpoint of apiculture. According to the ranking based on fructose content, which considers human sensation of taste, the most favourable cultivars were 'Ujfehertoi furtos', 'Meteor USA' and 'Korai pipacs'. Based on total sugar content the secretory products preferred by bees were those of 'Ujfehertoi furtos' (in three seasons); 'Korai pipacs', 'Erdi nagygyumolcsű', 'Sarandi S/Gy', 'Deb...

Summary Objective To demonstrate the capability of a clinical 3 T human scanner in performing quantitative MR experiments in the rat brain. Material and methods In vivo, measurements on eight Wistar rats were performed. Longitudinal relaxation time (T1) and transverse relaxation time (T2) measurements were set up at a spatial resolution of 0.3 × 0.3 × 1 mm3. Diffusion-weighted imaging was also applied and the evaluation included both mono- and biexponential approaches (b-value up to 6000 s/mm2). Besides quantitative imaging, the rat brain was also scanned at a microscopic resolution of 130 × 130 × 130 μm3. Quantitative proton spectroscopy was also carried out on the rat brain with water as internal reference. Results T1 and T2 for the rat brain cortex were 1272 ± 85 ms and 75 ± 2 ms, respectively. Diffusion-weighted imaging yielded accurate diffusion coefficient measurements at both low and high b-value ranges. The concentrations of MR visible metabolites were determined for the major resonances (i.e., N-acetyl-aspartate, choline and creatine) with acceptable accuracy. Conclusion The results suggest that quantitative imaging and spectroscopy can be carried out on small animals on high-field clinical scanners.

Background : Based on circumstantial evidences, we (Gallyas, F., Farkas, O., Mazlo, M., 2004. Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of “dark” (compacted) neurons. Biol. Cell 96, 313–324.) proposed that the formation of “dark” neurons (striking compaction of visibly normal ultrastructural elements accompanied with large-scale fluid excretion), which occur in many neurological diseases such as ischemia, proceeds with a non-enzymatic mechanism. Objective: To support this proposition, the present paper deals with the results of supravital experiments using microwave irradiation. Method: After transcardial glutaraldehyde fixation followed by decapitation, a pin was stuck into rat brains just before and just after they were warmed up to 80 °C and maintained at this temperature for various periods of time by controlled microwave irradiation. Results: Independently of the duration of irradiation, the pre-irradiation pin sticking produced numerous “dark” neurons in an approximating 500-μm-wide zone around its track whereas the post-irradiation pin sticking did the same only when the irradiation was shorter than 55 s. The excreted fluid was present in neighbouring astrocytic processes but not in the extracellular space. Conclusions: The formation of “dark” neurons is completed in less than 55 s under the circumstances of the experiment. As neurons are poor in readily consumable chemical energy in the absence of blood circulation, this rapid and massive fluid excretion cannot be explained by any enzyme-mediated membrane-related pump mechanism. An osmotic mechanism can also be discounted. In contrast, it is in conformity with the above mentioned non-enzymatic (physicochemical) phenomenon, the phase transition of a supra-cytoskeletal gel network storing free energy in the form of non-covalent interactions.

Several in vitro and in vivo experiments have demonstrated the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in focal cerebral ischemia, Parkinson's disease and traumatic brain injury (TBI). The aim of the present study was to analyze the effect of PACAP administration on diffuse axonal injury (DAI), an important contributor to morbidity and mortality associated with TBI, in a central fluid percussion (CFP) model of TBI. Rats were subjected to moderate (2 Atm) CFP injury. Thirty min after injury, 100 microg PACAP was administered intracerebroventricularly. DAI was assessed by immunohistochemical detection of beta-amyloid precursor protein, indicating impaired axoplasmic transport, and RMO-14 antibody, representing foci of cytoskeletal alterations (neurofilament compaction), both considered classical markers of axonal damage. Analysis of damaged, immunoreactive axonal profiles revealed significant axonal protection in the PACAP-treated versus vehicle-treated animals in the corticospinal tract, as far as traumatically induced disturbance of axoplasmic transport and cytoskeletal alteration were considered. Similarly to our former observations in an impact acceleration model of diffuse TBI, the present study demonstrated that PACAP also inhibits DAI in the CFP injury model. The finding indicates that PACAP and derivates can be considered potential candidates for further experimental studies, or purportedly for clinical trials in the therapy of TBI.

Traumatic brain injury (TBI) represents a leading cause of death in western countries. Despite all research efforts we still lack any pharmacological agent that could effectively be utilized in the clinical treatment of TBI. Detailed unraveling of the pathobiological processes initiated by/operant in TBI is a prerequisite to the development of rational therapeutic interventions. In this review we provide a summary of those therapeutic interventions purported to inhibit the cell death (CD) cascades ignited in TBI. On noxious stimuli three major forms of CD, apoptosis, autophagia and necrosis may occur. Apoptosis can be induced either via the mitochondrial (intrinsic) or the receptor mediated (extrinsic) pathway; endoplasmic reticular stress is the third trigger of caspase-mediated apoptotic processes. Although, theoretically pan-caspase inhibition could be an efficient tool to limit apoptosis and thereby the extent of TBI, potential cross-talk between various avenues of CD suggests that more upstream events, particularly the preservation of the cellular energy homeostasis (cyclosporine-A, poly ADP ribose polymerase (PARP) inhibition, hypothermia treatment) may represent more efficient therapeutic targets hopefully also translated to the clinical care of the severely head injured.

Floral activity was studied in two apple cultivars: an Erwinia-tolerant (Treedorn') and a sensitive one (`Sampion'). Since more types of protogyny occur in apples, the period of stigma activity is different. Papillae of the exposed stigma in flowers of 'Freedom' function longer (usually more than a week) than in the delayed homogamous `Sampion'. Despite of this, cv. 'Freedom' is tolerant to Erwinia amylovora (Burr.) Winslow et al., suggesting no relationship between the floral biological type (including the exposure and longevity of stigma) and the infection by E. amylovora. According to SEM micrographs, nectary stomata in `Freedom' are already open in the flower bud, where nectar secretion starts and continues until the senescence of the stigma. However, the long period of nectar secretion does not create optimal conditions for bacterial growth, since nectar production is scant in the flowers of 'Freedom'. The surface of the nectary, its nectar-retaining capacity, and the amount and concentration of nectar may influence the susceptibility of apple cultivars. It is manifested well by the smooth nectary surface with nectary stomata rising slightly above the epidermis in flowers of cv. 'Freedom', contrasting the wrinkled, striate nectary surface with slightly sunken stomata in the flowers of 'Sampion'.

The composition of floral nectar in sour cherry cultivars studied in 1997 at Újfehértó was in agreement with our previous data, the three most frequent sugar components being glucose, fructose and sucrose. Nectar secreted at night is generally more diluted than nectar produced during the day. None of the nectar samples produced at night reached the threshold value (100 mg/ml) of bee visitation. In the majority of cultivars the difference in concentration between night and day nectar is not too high, but in two cultivars, 'Korai pipacs' and ‘Újfehértói fürtös’ the difference is quite significant. Most sucrose was found in the nectar of cvs. 'Érdi jubileum' and ‘Újfehértói fürtös’, but a high amount of sucrose was measured also in the flowers of 'Korai pipacs' and 'Meteor USA'. Nectar concentration varies from opening of the flower to petal fall to a smaller or greater degree, depending on the given cultivar. From the 9 sour cherry cultivars studied ‘Újfehértói fürtös’ and Tandy 48' had the most attractive nectar for bees. There was no correlation between nectar composition and free fertilisation. On the basis of nectar composition the majority of the sour cherry cultivars studied can be classified into the sucrose-rich category; only one cultivar, 'Érdi jubileum' had a sucrose-dominant secretory product. The composition of nectar in the studied sour cherry cultivars is preferred by bees.

Kherani and Auer [6] recently reported a study in Acta Neuropathologica relating to the mechanism of the formation of “dark” neurons. Their observations led them to suggest that (1) both in vivo and during the early postmortem period, glutamate release and transmembrane ion Xuxes cause some perturbation in a proportion of neurons, which become shrunken, hyperchromatic under the light microscope, and condensed, hyperelectron-dense at the ultrastructural level, but only while aldehyde Wxation is underway, (2) the shrinkage/condensation is executed by transmembrane ion Xuxes or by actin Wlaments, and (3) everything considered, the formation of “dark” neurons is a biotic process. This idea was logically deduced in part from the results of an experiment dealing with the formation of the artefactual “dark” neurons of Cammermeyer (i.e., neurons formed even in normal brain tissue when removed from the skull without previous transcardial aldehyde Wxation followed by a 24-h delay [1]), and in part from the earlier experience of neuropathologists that artefactual “dark” neurons are generally not encountered either in autopsy materials when the postmortem period is long or after nonaldehyde Wxation. Furthermore, they assumed that this idea also applies to the nonartefactual “dark” neurons produced in vivo by ischemia, hypoglycemia, or epilepsy. In a number of communications (reviewed by [3]), our team have postulated another mechanism, which unfortunately is not mentioned in their study. This mechanism postulates that, in each neuron, the intracellular spaces among the ultrastructural elements of conventional transmission electron microscopy are Wlled with a continuous trabecular gel consisting of proteins, ions and water; this gel stores noncovalent (mechanical) free energy, at the expense of which a volume-shrinking phase transition can spread throughout its whole soma-dendrite domain in response to a chemical or a physical noxa (for the existence and importance of such gels in cell biology, see [10]). The pivotal observations supporting this mechanism were as follows: (1) if initiated by physical (nonbiotic) noxae, the formation of “dark”-neurons took place even under conditions that were extremely unfavorable for enzyme-mediated (biotic) processes [4, 7, 9], (2) independently of the nature (physical or chemical) of the initiating noxa and of the circumstances (favorable or unfavorable for enzymatic processes) of the “dark”-neuron formation, the excess water appeared in neighboring astrocytic elements, but not in the surrounding extracellular space [2–9] (the “destination” of transmembrane ion Xuxes), and (3) in each in-vivo case we investigated [2, 5, 8], both the recovering and the dying “dark” neurons displayed ultrastructural signs proving indisputably that they had been compact and electron-dense even long before aldehyde Wxation. To obtain direct evidence against the role of aldehyde Wxation in the formation of the nonartefactual “dark” neurons, we produced “dark” granule neurons in the hippocampal dentate gyrus of four anesthetized living rats via a single condenser discharge, and in the neocortex of four other anesthetized living rats via a pin puncture head injury (for the methodological details and the animal care, see [2]). In each group, two rats were immediately Wxed by the transcardial perfusion of 500 ml of a 3:1 mixture of methanol and chloroform, while the cut-oV heads of the other rats were subjected to Wxation by microwave irradiation (continuous 750 W, controlled 80°C, 1 min). The brains were removed from the skull 1 day later [1] and processed (without aldehyde postWxation) for electron microscopy as usual. P. Bukovics · J. Pal · F. Gallyas (&) Department of Neurosurgery, University of Pecs, 7623 Ret utca 2, Pecs, Hungary e-mail: ferenc.gallyas.sen@aok.pte.hu