The Spinal Cord Damage in a Rat Asphyxial Cardiac Arrest/Resuscitation Model.

BACKGROUND: After cardiac arrest/resuscitation (CA/R), animals often had massive functional restrictions including spastic paralysis of hind legs, disturbed balance and reflex abnormalities. Patients who have survived CA also develop movement restrictions/disorders. A successful therapy requires detailed knowledge of the intrinsic damage pattern and the respective mechanisms. Beside neurodegenerations in the cerebellum and cortex, neuronal loss in the spinal cord could be a further origin of such movement artifacts. METHODS: Thus, we aimed to evaluate the CA/R-induced degeneration pattern of the lumbar medulla spinalis by immunocytochemical expression of SMI 311 (marker of neuronal perikarya and dendrites), IBA1 (microglia marker), GFAP (marker of astroglia), calbindin D28k (marker of the cellular neuroprotective calcium-buffering system), MnSOD (neuroprotective antioxidant), the transcription factor PPARγ and the mitochondrial marker protein PDH after survival times of 7 and 21 days. The CA/R specimens were compared with those from sham-operated and completely naïve rats. RESULTS & CONCLUSION: The main ACA/R-mediated results were: (1) degeneration of lumbar spinal cord motor neurons, characterized by neuronal pyknotization and peri-neuronal tissue artifacts; (2) attendant activation of microglia in the short-term group; (3) attendant activation of astroglia in the long-term group; (4) degenerative pattern in the intermediate gray matter; (5) activation of the endogenous anti-oxidative defense systems calbindin D28k and MnSOD; (6) activation of the transcription factor PPARγ, especially in glial cells of the gray matter penumbra; and (7) activation of mitochondria. Moreover, marginal signs of anesthesia-induced cell stress were already evident in sham animals when compared with completely naïve spinal cords. A correlation between the NDS and the motor neuronal loss could not be verified. Thus, the NDS appears to be unsuitable as prognostic tool.

Normoxic post-ROSC ventilation delays hippocampal CA1 neurodegeneration in a rat cardiac arrest model, but does not prevent it.

The European Resuscitation Guidelines recommend that survivors of cardiac arrest (CA) be resuscitated with 100% O2 and undergo subsequent-post-return of spontaneous circulation (ROSC)-reduction of O2 supply to prevent hyperoxia. Hyperoxia produces a "second neurotoxic hit," which, together with the initial ischemic insult, causes ischemia-reperfusion injury. However, heterogeneous results from animal studies suggest that normoxia can also be detrimental. One clear reason for these inconsistent results is the considerable heterogeneity of the models used. In this study, the histological outcome of the hippocampal CA1 region following resuscitation with 100% O2 combined with different post-ROSC ventilation regimes (21%, 50%, and 100% O2) was investigated in a rat CA/resuscitation model with survival times of 7 and 21 days. Immunohistochemical stainings of NeuN, MAP2, GFAP, and IBA1 revealed a neuroprotective potency of post-ROSC ventilation with 21% O2, although it was only temporary. This limitation should be because of the post-ROSC intervention targeting only processes of ischemia-induced secondary injury. There were no ventilation-dependent effects on either microglial activation, reduction of which is accepted as being neuroprotective, or astroglial activation, which is accepted as being able to enhance neurons' resistance to ischemia/reperfusion injury. Furthermore, our findings verify the limited comparability of animal studies because of the individual heterogeneity of the animals, experimental regimes, and evaluation procedures used.

Binding varicella zoster virus: an underestimated facet of insulin-degrading enzyme´s implication for Alzheimer´s disease pathology?

We shortly discuss a possible contribution of insulin-degrading enzyme to Alzheimer´s disease pathology by binding varicella zoster virus glycoprotein E, which increases the infectivity and cell-cell spread of the virus.

Relative Resilience of Cerebellar Purkinje Cells in a Cardiac Arrest/Resuscitation Rat Model.

BACKGROUND: In studies on cardiac arrest (CA)/resuscitation (R) injury, Purkinje cell degeneration was described, however, with inconsistent data concerning severity and time point of manifestation. Moreover, CA/R studies paid only limited attention to inhibitory stellate interneurons. To this aim, the hypothesis that cerebellar could be relatively resilient toward CA/R because of diverse cellular defense mechanisms including interaction with stellate cells was tested. METHODS: We examined rats with survival times of 6, 24, and 48 h, and 7 and 21 days in comparison with sham- and nonoperated animals. Thereby, we focused on the immunohistochemical expression of cfos, MnSOD, Bcl2, caspase 3, parvalbumin, calbindin D28 k, MAP2, IBA1, and GFAP, especially in the particular sensitivity to CA/R cerebellar lobule IX. Hippocampal CA1 degeneration was demonstrated by expression patterns of MAP2 and NeuN in combination with IBA1 and GFAP. RESULTS/CONCLUSIONS: Comparative analysis of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells confirmed a relative resil-ience of Purkinje cells to CA/R. We found only a notable degeneration of Purkinje cell neuronal fiber network, which, however, not necessarily led to neuronal cell death. To induce significant Purkinje cell loss, a stronger ischemic trigger seems to be needed. As possible Purkinje cell-protecting mechanisms, we would propose: (1) activation of inhibitory stellate cells, shown by cfos, MnSOD, and Bcl2 expression, balancing out ischemia-induced excitation and inhibition of Purkinje cells; (2) translocation of the calcium-buffering system, shown by parvalbumin and calbindin D28 k expression, protecting Purkinje cells from detrimental calcium overload; (3) activation of the neuron-astrocyte cross talk, protecting Purkinje cells from over-excitation by removing potassium and neurotransmitters from the extracellular space; (4) activation of the effective and long-lasting MnSOD defense system; and (5) of the anti-apoptotic protein Bcl2 in Purkinje cells itself. Moreover, the results emphasize the limited comparability of animal CA/R studies because of the heterogeneity of the used experimental regimes.

The hypothalamus and neuropsychiatric disorders: psychiatry meets microscopy.

The past decades have witnessed an explosion of knowledge on brain structural abnormalities in schizophrenia and depression. Focusing on the hypothalamus, we try to show how postmortem brain microscopy has contributed to our understanding of mental disease-related pathologic alterations of this brain region. Gross anatomical abnormalities (volume changes of the third ventricle, the hypothalamus, and its nuclei) and alterations at the cellular level (loss of neurons, increased or decreased expression of hypothalamic peptides such as oxytocin, vasopressin, corticotropin-releasing hormone, and other regulatory factors as well as of enzymes involved in neurotransmitter and neuropeptide metabolism) have been reported in schizophrenia and/or depression. While histologic research has mainly concentrated on neurons, little is currently known about the impact of non-neuronal cells for hypothalamus pathology in mental disorders. Their study would be a rewarding task for the future.

The expression of the MSC-marker CD73 and of NF2/Merlin are correlated in meningiomas.

Mesenchymal stem cells (MSC) have been found in various cancers and were discussed to influence tumor biology. Cells fulfilling the complete MSC criteria, including surface marker expression (CD73, CD90, CD105) and tri-lineage differentiation, have been isolated solely from a low percentage of high-grade meningiomas. In contrast, pure co-expression of the surface-markers was relatively frequent, raising the question for an additional role of these membrane proteins in meningiomas. Therefore, here we analyzed the expression of CD73, CD90 and CD105 in a series of meningiomas of all grades. Although no significant association of any marker with meningeal tumor growth per se or with tumor-grade was observed, we detected a positive Pearson correlation (r = 0.55, p ≤ 0.05) in low-grade tumors between CD73 and the most relevant tumor suppressor NF2/Merlin, supported by a tendency of lower CD73 expression in cases with allelic losses at the NF2-locus, which express significantly lower NF2/Merlin-mRNA (p ≤ 0.05). In two pairs of syngenous meningeal or meningioma cell lines with or without shRNA-mediated knockdown of NF2/Merlin a nearly complete loss of CD73 mRNA expression was observed after the knockdown (p ≤ 0.001). This suggested that the correlation observed in tumors may result from a direct functional link between Merlin and CD73. Since CD73 is a 5'-exonucleotidase (termed NT5E), we discuss a potential role of NT5E-mediated purinergic signaling to modulate actin-cytoskeleton and cell contacts, which may be a functional link to NF2/Merlin.

Insulin-regulated aminopeptidase immunoreactivity is abundantly present in human hypothalamus and posterior pituitary gland, with reduced expression in paraventricular and suprachiasmatic neurons in chronic schizophrenia.

The vasopressin- and oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP) is expressed in various organs including the brain. However, knowledge about its presence in human hypothalamus is fragmentary. Functionally, for a number of reasons (genetic linkage, hydrolysis of oxytocin and vasopressin, its role as angiotensin IV receptor in learning and memory and others) IRAP might play a role in schizophrenia. We studied the regional and cellular localization of IRAP in normal human brain with special emphasis on the hypothalamus and determined numerical densities of IRAP-expressing cells in the paraventricular, supraoptic and suprachiasmatic nuclei in schizophrenia patients and controls. By using immunohistochemistry and Western blot analysis, IRAP was immunolocalized in postmortem human brains. Cell countings were performed to estimate numbers and numerical densities of IRAP immunoreactive hypothalamic neurons in schizophrenia patients and control cases. Shape, size and regional distribution of IRAP-expressing cells, as well the lack of co-localization with the glia marker glutamine synthetase, show that IRAP is expressed in neurons. IRAP immunoreactive cells were observed in the hippocampal formation, cerebral cortex, thalamus, amygdala and, abundantly, hypothalamus. Double labeling experiments (IRAP and oxytocin/neurophysin 1, IRAP with vasopressin/neurophysin 2) revealed that IRAP is present in oxytocinergic and in vasopressinergic neurons. In schizophrenia patients, the numerical density of IRAP-expressing neurons in the paraventricular and the suprachiasmatic nuclei is significantly reduced, which might be associated with the reduction in neurophysin-containing neurons in these nuclei in schizophrenia. The pathophysiological role of lowered hypothalamic IRAP expression in schizophrenia remains to be established.

In human brain ornithine transcarbamylase (OTC) immunoreactivity is strongly expressed in a small number of nitrergic neurons.

There is recent evidence for ornithine transcarbamylase (OTC) expression in adult human brain. We decided to immunocytochemically map OTC throughout brain, and to further characterize OTC-immunopositive neurons. By using double immunolabeling technique for OTC and neuronal nitric oxide synthase (nNOS) OTC protein expression was revealed in a small subset of nitrergic (nNOS) neurons. Since citrulline (the reaction product of OTC) enhances the bioavailability of L-arginine, the substrate of nNOS, it is conceivable that OTC activity supports NO production in nitrergic neurons.

Sciatic nerve ligation causes impairment of mitochondria associated with changes in distribution, respiration, and cardiolipin composition in related spinal cord neurons in rats.

Sciatic nerve irritation is often associated with disturbed Ca(2+) homeostasis in related neurons of the spinal cord. Since mitochondria substantially contribute to Ca(2+) homeostasis and little information is available, we studied the effects of loose sciatic nerve ligation, a chronic constriction injury (CCI), on neuronal mitochondria of the L3-L6 regions. Three groups of rats (untreated, sham operated, and ligated) were explored. For the characterization of mitochondria, specimens of the L3-L6 spinal cord regions were evaluated with respect to intracellular localization using pyruvate dehydrogenase immunohistochemistry and Mitotracker Red, and the ATP producing machinery by LC-MS/MS technique for the analysis of cardiolipin and high-resolution respirometry for the measurement of oxygen consumption. Therefore, the phospholipid cardiolipin supports electron transfer within the respiratory chain as part of mitochondrial respiration and is of high impact on the physical properties of the mitochondrial membrane system. Histological analysis of spinal cord motor neurons revealed clustering of mitochondria in ipsilateral samples from ligated animals 14 days after the insult. This phenomenon was similarly evident in the respective contralateral side. The intensity of MT-Red staining was enhanced exclusively at the ipsilateral side, indicating increased mitochondrial activity. CCI of the sciatic nerve caused massive changes in the composition of cardiolipin reflecting mitochondrial impairment in the early phase followed by regeneration processes as late response. Sciatic nerve CCI caused decrease in the capacity of mitochondrial ATP production that recovered within 14 days after treatment. In conclusion, we provide evidence that clustering of mitochondria, already verified for the spinal cord sensory neurons after CCI, also occurs in the respective motor neurons. Further we have demonstrated transient impairment of the capacity of mitochondrial ATP production in tissue samples. Stress-dependent changes in cardiolipin composition are sensitive markers and mediators of the response process including impairment and regeneration.

Possible sources and functions of L-homoarginine in the brain: review of the literature and own findings.

L-Homoarginine is a cationic amino acid derivative, which is structurally related to L-arginine and lysine. Several lines of evidence point to nervous tissue as an important target of homoarginine action. In the mammalian brain homoarginine can be detected in noticeable quantities, but its origin is currently poorly explored. In part I of this review we try to show that both uptake and transport into brain (carried out by cationic amino acid transporters) and local synthesis in the brain (carried out by the homoarginine-synthesizing enzymes L-arginine:glycine amidinotransferase and ornithine transcarbamylse) might contribute to homoarginine brain content. We then give a brief overview about the multiple effects of homoarginine on the healthy brain and show that both homoarginine excess and deficiency are potentially harmful to the central nervous system. In part II, we shortly report about own experiments with regard to the cellular localization of cationic amino acid transporters, as well the enzymes L-arginine:glycine amidinotransferase and ornithine transcarbamylse, in human and rat brains.

Lipidomic analysis of molecular cardiolipin species in livers exposed to ischemia/reperfusion.

Transient hepatic ischemia can cause significant liver injury. A central and early event in ischemia/reperfusion (I/R) injury is the impairment of mitochondria. The phospholipid cardiolipin (CL) is required for efficient mitochondrial function. The aim of this study was to analyze composition, content, and oxidation of CL in dependence of I/R stress. Therefore, we exposed rat livers to 20 min ischemia by interrupting the perfusion with Krebs-Ringer solution in situ. Tissue histology as well as increased activities of LDH, GLDH, and ASAT analysed in the efflux after 50 min reperfusion indicated impairment of the liver. For the analysis of local CL distribution the liver homogenate was separated according to density into 11 fractions. The fractions displayed different contents of CL and citrate synthase peaking at density of about 1.07 g/cm(3). Among the fractions, the distribution of molecular CL species significantly differed. I/R caused loss of about 30 % CL and 17 % citrate synthase activity. Further, I/R shifted the CL and citrate synthase activity profile toward lower densities. Oxidized CL was exclusively found in fractions with high CL and citrate synthase content after I/R stress. I/R treatment caused significant changes in the distribution of molecular CL species. Our data demonstrate that I/R causes significant decrease in CL content and increase of oxidized CL that may be of impact for impairment of mitochondrial function by I/R. These results lead to the suggestion that strategies supporting anti-oxidative defence and CL synthesis may be beneficial to reduce I/R injury of the liver.

Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries.

Oxidation of cardiolipin is involved in functional impairment and disintegration of liver mitochondria by hypoxia/reoxygenation in the presence of increased Ca²âº concentrations.

The aim of this study was to investigate the interrelationship between the mitochondrial phospholipid cardiolipin (CL), mitochondrial respiration and morphology in dependence on hypoxia/reoxygenation and Ca(2+). Therefore, we subjected rat liver mitochondria to hypoxia/reoxygenation at different extramitochondrial Ca(2+) concentrations and analysed mitochondrial respiration, morphology, CL content, the composition of molecular CL species, oxidation of CL and two mono-lyso-CL species. Hypoxia/reoxygenation in the presence of elevated extramitochondrial Ca(2+) concentration caused dramatic impairment of mitochondrial respiration and morphology. Concomitantly, increased amounts of oxidised CL were detected in the incubation medium after the treatment. Hypoxia/reoxygenation alone caused degradation of CL. The treatments had no effect on the composition of molecular CL species. Our data support the hypothesis that CL oxidation and CL degradation are involved in mitochondrial injury caused by hypoxia/reoxygenation and Ca(2+). Our results further suggest that prevention of CL oxidation by modification of CL composition may support the beneficial action of antioxidants during hypoxia/reoxygenation in the presence of elevated Ca(2+) concentrations.

Wide distribution of CREM immunoreactivity in adult and fetal human brain, with an increased expression in dentate gyrus neurons of Alzheimer's as compared to normal aging brains.

Human cyclic AMP response modulator proteins (CREMs) are encoded by the CREM gene, which generates 30 or more different CREM protein isoforms. They are members of the leucine zipper protein superfamily of nuclear transcription factors. CREM proteins are known to be implicated in a plethora of important cellular processes within the CNS. Amazingly, little is known about their cellular and regional distribution in the brain, however. Therefore, we studied by means of immunohistochemistry and Western blotting the expression patterns of CREM in developing and adult human brain, as well as in brains of Alzheimer's disease patients. CREM immunoreactivity was found to be widely but unevenly distributed in the adult human brain. Its localization was confined to neurons. In immature human brains, CREM multiple neuroblasts and radial glia cells expressed CREM. In Alzheimer's brain, we found an increased cellular expression of CREM in dentate gyrus neurons as compared to controls. We discuss our results with regard to the putative roles of CREM in brain development and in cognition.

The many facets of CD26/dipeptidyl peptidase 4 and its inhibitors in disorders of the CNS - a critical overview.

Dipeptidyl peptidase 4 is a serine protease that cleaves X-proline or X-alanine in the penultimate position. Natural substrates of the enzyme are glucagon-like peptide-1, glucagon inhibiting peptide, glucagon, neuropeptide Y, secretin, substance P, pituitary adenylate cyclase-activating polypeptide, endorphins, endomorphins, brain natriuretic peptide, beta-melanocyte stimulating hormone and amyloid peptides as well as some cytokines and chemokines. The enzyme is involved in the maintenance of blood glucose homeostasis and regulation of the immune system. It is expressed in many organs including the brain. DPP4 activity may be effectively depressed by DPP4 inhibitors. Apart from enzyme activity, DPP4 acts as a cell surface (co)receptor, associates with adeosine deaminase, interacts with extracellular matrix, and controls cell migration and differentiation. This review aims at revealing the impact of DPP4 and DPP4 inhibitors for several brain diseases (virus infections affecting the brain, tumours of the CNS, neurological and psychiatric disorders). Special emphasis is given to a possible involvement of DPP4 expressed in the brain.While prominent contributions of extracerebral DPP4 are evident for a majority of diseases discussed herein; a possible role of "brain" DPP4 is restricted to brain cancers and Alzheimer disease. For a number of diseases (Covid-19 infection, type 2 diabetes, Alzheimer disease, vascular dementia, Parkinson disease, Huntington disease, multiple sclerosis, stroke, and epilepsy), use of DPP4 inhibitors has been shown to have a disease-mitigating effect. However, these beneficial effects should mostly be attributed to the depression of "peripheral" DPP4, since currently used DPP4 inhibitors are not able to pass through the intact blood-brain barrier.

The many "Neurofaces" of Prohibitins 1 and 2: Crucial for the healthy brain, dysregulated in numerous brain disorders.

Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are proteins that are nearly ubiquitously expressed. They are localized in mitochondria, cytosol and cell nuclei. In the healthy CNS, they occur in neurons and non-neuronal cells (oligodendrocytes, astrocytes, microglia, and endothelial cells) and fulfill pivotal functions in brain development and aging, the regulation of brain metabolism, maintenance of structural integrity, synapse formation, aminoacidergic neurotransmission and, probably, regulation of brain action of certain hypothalamic-pituitary hormones.With regard to the diseased brain there is increasing evidence that prohibitins are prominently involved in numerous major diseases of the CNS, which are summarized and discussed in the present review (brain tumors, neurotropic viruses, Alzheimer disease, Down syndrome, Fronto-temporal and vascular dementia, dementia with Lewy bodies, Parkinson disease, Huntington disease, Multiple sclerosis, Amyotrophic lateral sclerosis, stroke, alcohol use disorder, schizophrenia and autism). Unfortunately, there is no PHB-targeted therapy available for any of these diseases.

Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia.

BACKGROUND: Magnetic nanoparticles (MNPs) offer a large range of applications in life sciences. Applications in neurosciences are one focus of interest. Unfortunately, not all groups have access to nanoparticles or the possibility to develop and produce them for their applications. Hence, they have to focus on commercially available particles. Little is known about the uptake of nanoparticles in primary cells. Previously studies mostly reported cellular uptake in cell lines. Here we present a systematic study on the uptake of magnetic nanoparticles (MNPs) by primary cells of the nervous system. RESULTS: We assessed the internalization in different cell types with confocal and electron microscopy. The analysis confirmed the uptake of MNPs in the cells, probably with endocytotic mechanisms. Furthermore, we compared the uptake in PC12 cells, a rat pheochromocytoma cell line, which is often used as a neuronal cell model, with primary neuronal cells. It was found that the percentage of PC12 cells loaded with MNPs was significantly higher than for neurons. Uptake studies in primary mixed neuronal/glial cultures revealed predominant uptake of MNPs by microglia and an increase in their number. The number of astroglia and oligodendroglia which incorporated MNPs was lower and stable. Primary mixed Schwann cell/fibroblast cultures showed similar MNP uptake of both cell types, but the Schwann cell number decreased after MNP incubation. Organotypic co-cultures of spinal cord slices and peripheral nerve grafts resembled the results of the dispersed primary cell cultures. CONCLUSIONS: The commercial MNPs used activated microglial phagocytosis in both disperse and organotypic culture systems. It can be assumed that in vivo application would induce immune system reactivity, too. Because of this, their usefulness for in vivo neuroscientific implementations can be questioned. Future studies will need to overcome this issue with the use of cell-specific targeting strategies. Additionally, we found that PC12 cells took up significantly more MNPs than primary neurons. This difference indicates that PC12 cells are not a suitable model for natural neuronal uptake of nanoparticles and qualify previous results in PC12 cells.

Effects of risperidone treatment in adolescence on hippocampal neurogenesis, parvalbumin expression, and vascularization following prenatal immune activation in rats.

Maternal infection in pregnancy is an environmental risk factor for the development of schizophrenia and related disorders in the offspring, and this association is recapitulated in animal models using gestational infection or immune stimulation. We have recently shown that behavioral abnormalities and altered hippocampal morphology emerging in adult offspring of dams treated with the viral mimic polyriboinosinic-polyribocytidilic acid (poly I:C) are prevented by treatment with the atypical antipsychotic drug risperidone (RIS) in adolescence. Here we used a battery of cellular markers and Nissl stain to morphometrically analyze different hippocampal cell populations in the offspring of poly I:C and saline-treated mothers that received saline or RIS in adolescence, at different time points of postnatal development. We report that impaired neurogenesis, disturbed micro-vascularization and loss of parvalbumin-expressing hippocampal interneurons, are found in the offspring of poly I:C-treated dams. Most, but not all, of these neuropathological changes are not present in poly I:C offspring that had been treated with RIS. These effects may be part of the complex processes underlying the capacity of RIS treatment in adolescence to prevent structural and behavioral abnormalities deficits in the poly I:C offspring.