Katrin Frauenknecht

Citicoline Enhances Neuroregenerative Processes After Experimental Stroke in Rats

Background and Purpose— The neuroprotective potential of citicoline in acute ischemic stroke has been shown in many experimental studies and, although the exact mechanisms are still unknown, a clinical Phase III trial is currently underway. Our present study was designed to check whether citicoline also enhances neuroregeneration after experimental stroke. Methods— Forty Wistar rats were subjected to photothrombotic stroke and treated either with daily injections of citicoline (100 mg/kg) or vehicle for 10 consecutive days starting 24 hours after ischemia induction. Sensorimotor tests were performed after an adequate training period at Days 1, 10, 21, and 28 after stroke. Then brains were removed and analyzed for infarct size, glial scar formation, neurogenesis, and ligand binding densities of excitatory and inhibitory neurotransmitter receptors. Results— Animals treated with citicoline showed a significantly better neurological outcome at Days 10, 21, and 28 after ischemia, which could not be attributed to differences in infarct volumes or glial scar formation. However, neurogenesis in the dentate gyrus, subventricular zone, and peri-infarct area was significantly increased by citicoline. Furthermore, enhanced neurological outcome after citicoline treatment was associated with a shift toward excitation in the perilesional cortex. Conclusions— Our present data demonstrate that, apart from the well-known neuroprotective effects in acute ischemic stroke, citicoline also possesses a substantial neuroregenerative potential. Thanks to its multimodal effects, easy applicability, and history as a well-tolerated drug, promising possibilities of neurological treatment including chronic stroke open up.

Propofol impairs neurogenesis and neurologic recovery and increases mortality rate in adult rats after traumatic brain injury.

Objective:Limited data are available on the influence of sedation for critical care therapy with the widely used anesthetic propofol on recovery from acute traumatic brain injury. To establish the influence of propofol on endogenous neurogenesis and functional recovery after traumatic brain injury, rats were sedated with propofol either during or 2 hours after experimental traumatic brain injury. Design:Randomized controlled animal study. Setting:University research laboratory. Subjects:One hundred sixteen male Sprague Dawley rats. Interventions:Mechanical brain lesion by controlled cortical impact. Measurements and Main Results:This study investigated the dose-dependent influence of propofol (36 or 72u2009mg/kg/hr) either during controlled cortical impact induction or in a delayed application protocol 2 hours after experimental traumatic brain injury. Infusion of propofol resulted in 1) aggravation of neurologic dysfunction, 2) increased 28-day mortality rate, and 3) impaired posttraumatic neurogenesis (5-bromo-2-deoxyuridine + NeuN-positive cells). Application of propofol during trauma induction afforded a significant stronger effect in the high-dose group compared with low-dose propofol. In the posttrauma protocol, animals were sedated with sevoflurane during the controlled cortical impact injury, and propofol was given after an awake phase. In these animals, propofol increased mortality rate and impaired neurologic function and neurogenesis compared with animals without delayed propofol anesthesia. Conclusions:The results show that propofol may prevent or limit reparative processes in the early-phase postinjury. The results therefore indicate that anesthetics may be potentially harmful not only in very young mammalians but also in adult animals following acute cerebral injuries. The results provide first evidence for an altered sensitivity for anesthesia-related negative effects on neurogenesis, functional outcome, and survival in adult rats with brain lesions.

Systemic PaO2 Oscillations Cause Mild Brain Injury in a Pig Model.

Objective:Systemic PaO2 oscillations occur during cyclic recruitment and derecruitment of atelectasis in acute respiratory failure and might harm brain tissue integrity. Design:Controlled animal study. Setting:University research laboratory. Subjects:Adult anesthetized pigs. Interventions:Pigs were randomized to a control group (anesthesia and extracorporeal circulation for 20u2009hr with constant PaO2, n = 10) or an oscillation group (anesthesia and extracorporeal circulation for 20u2009hr with artificial PaO2 oscillations [3 cycles min–1], n = 10). Five additional animals served as native group (n = 5). Measurements and Main Results:Outcome following exposure to artificial PaO2 oscillations compared with constant PaO2 levels was measured using 1) immunohistochemistry, 2) real-time polymerase chain reaction for inflammatory markers, 3) receptor autoradiography, and 4) transcriptome analysis in the hippocampus. Our study shows that PaO2 oscillations are transmitted to brain tissue as detected by novel ultrarapid oxygen sensing technology. PaO2 oscillations cause significant decrease in NISSL-stained neurons (p < 0.05) and induce inflammation (p < 0.05) in the hippocampus and a shift of the balance of hippocampal neurotransmitter receptor densities toward inhibition (p < 0.05). A pathway analysis suggests that cerebral immune and acute-phase response may play a role in mediating PaO2 oscillation–induced brain injury. Conclusions:Artificial PaO2 oscillations cause mild brain injury mediated by inflammatory pathways. Although artificial PaO2 oscillations and endogenous PaO2 oscillations in lung-diseased patients have different origins, it is likely that they share the same noxious effect on the brain. Therefore, PaO2 oscillations might represent a newly detected pathway potentially contributing to the crosstalk between acute lung and remote brain injury.

A single systemic transient hypotension induces long-term changes in rats' MRI parameters and behavior: relation to aging

Abstract Objectives and Aim: The aim of this work was to examine whether a single 20-minute systemic hypotension induces short- and long-term changes in 12- and 18-month-old rat brain. Methods: Age-related changes were determined after hemorrhagic hypotension over a 6-month period of investigation using holeboard testing, diffusion-weighted (DW) magnetic resonance imaging (MRI) and T2 and T2* relaxometry. Results: The results showed a short-term but significant reduction in the apparent diffusion coefficient (ADC) after hypotension, which was accompanied by an increase in T2 relaxation time and deterioration in rat locomotor activity and memory capacities. Thereafter, a transient pseudo-normalization was regained. However, a significant difference in ADC and T2 relaxation time between controls and rats with transient hypotension and in rat reference memory was noticeable at month 6 after injury. A significant negative linear correlation was detected between T2 relaxation time and reference memory capacity. With regard to the effect of cerebral aging, significant changes were found in hippocampal ADC, T2 and T2* relaxation times and in behavioral parameters in 18-month-old rats compared with 12-month-old animals. Discussion and Conclusion: It was demonstrated that even a single oligemic event induces long-term changes in rat brain. Increased T2 reflects memory reduction. In addition, it has been shown that DW-MRI technique allows a longitudinal in vivo assessment of short- and long-term changes in rat brain. Further MRI perfusion works and detailed histological investigations on brain structure are necessary to study whether MRI measurements adequately reflect structural changes in the brain.

Neurotoxic Antibodies against the Prion Protein Do Not Trigger Prion Replication.

Prions are the infectious agents causing transmissible spongiform encephalopathies (TSE), progressive, inexorably lethal neurological diseases. Antibodies targeting the globular domain (GD) of the cellular prion protein PrPC trigger a neurotoxic syndrome morphologically and molecularly similar to prion disease. This phenomenon raises the question whether such antibodies induce infectious prions de novo. Here we exposed cerebellar organotypic cultured slices (COCS) to the neurotoxic antibody, POM1. We then inoculated COCS homogenates into tga20 mice, which overexpress PrPC and are commonly utilized as sensitive indicators of prion infectivity. None of the mice inoculated with COCS-derived lysates developed any signs of disease, and all mice survived for at least 200 days post-inoculation. In contrast, all mice inoculated with bona fide prions succumbed to TSE after 55–95 days. Post-mortem analyses did not reveal any signs of prion pathology in mice inoculated with POM1-COCS lysates. Also, lysates from POM1-exposed COCS were unable to convert PrP by quaking. Hence, anti-GD antibodies do not catalyze the generation of prion infectivity. These data indicate that prion replication can be separated from prion toxicity, and suggest that anti-GD antibodies exert toxicity by acting downstream of prion replication.

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

Summary Multiple sclerosis is an autoimmune disease that is caused by the interplay of genetic, particularly the HLA-DR15 haplotype, and environmental risk factors. How these etiologic factors contribute to generating an autoreactive CD4+ T cell repertoire is not clear. Here, we demonstrate that self-reactivity, defined as “autoproliferation” of peripheral Th1 cells, is elevated in patients carrying the HLA-DR15 haplotype. Autoproliferation is mediated by memory B cells in a HLA-DR-dependent manner. Depletion of B cells in vitro and therapeutically in vivo by anti-CD20 effectively reduces T cell autoproliferation. T cell receptor deep sequencing showed that in vitro autoproliferating T cells are enriched for brain-homing T cells. Using an unbiased epitope discovery approach, we identified RASGRP2 as target autoantigen that is expressed in the brain and B cells. These findings will be instrumental to address important questions regarding pathogenic B-T cell interactions in multiple sclerosis and possibly also to develop novel therapies.

Lung injury does not aggravate mechanical ventilation-induced early cerebral inflammation or apoptosis in an animal model

Introduction The acute respiratory distress syndrome is not only associated with a high mortality, but also goes along with cognitive impairment in survivors. The cause for this cognitive impairment is still not clear. One possible mechanism could be cerebral inflammation as result of a “lung-brain-crosstalk”. Even mechanical ventilation itself can induce cerebral inflammation. We hypothesized, that an acute lung injury aggravates the cerebral inflammation induced by mechanical ventilation itself and leads to neuronal damage. Methods After approval of the institutional and state animal care committee 20 pigs were randomized to one of three groups: lung injury by central venous injection of oleic acid (n = 8), lung injury by bronchoalveolar lavage in combination with one hour of injurious ventilation (n = 8) or control (n = 6). Brain tissue of four native animals from a different study served as native group. For six hours all animals were ventilated with a tidal volume of 7 ml kg-1 and a scheme for positive end-expiratory pressure and inspired oxygen fraction, which was adapted from the ARDS network tables. Afterwards the animals were killed and the brains were harvested for histological (number of neurons and microglia) and molecular biologic (TNFalpha, IL-1beta, and IL-6) examinations. Results There was no difference in the number of neurons or microglia cells between the groups. TNFalpha was significantly higher in all groups compared to native (p < 0.05), IL-6 was only increased in the lavage group compared to native (p < 0.05), IL-1beta showed no difference between the groups. Discussion With our data we can confirm earlier results, that mechanical ventilation itself seems to trigger cerebral inflammation. This is not aggravated by acute lung injury, at least not within the first 6 hours after onset. Nevertheless, it seems too early to dismiss the idea of lung-injury induced cerebral inflammation, as 6 hours might be just not enough time to see any profound effect.

Itch suppression in mice and dogs by modulation of spinal α2 and α3GABAA receptors

Chronic itch is a highly debilitating condition affecting about 10% of the general population. The relay of itch signals is under tight control by inhibitory circuits of the spinal dorsal horn, which may offer a hitherto unexploited therapeutic opportunity. Here, we found that specific pharmacological targeting of inhibitory α2 and α3GABAA receptors reduces acute histaminergic and non-histaminergic itch in mice. Systemic treatment with an α2/α3GABAA receptor selective modulator alleviates also chronic itch in a mouse model of atopic dermatitis and in dogs sensitized to house dust mites, without inducing sedation, motor dysfunction, or loss of antipruritic activity after prolonged treatment. Transsynaptic circuit tracing, immunofluorescence, and electrophysiological experiments identify spinal α2 and α3GABAA receptors as likely molecular targets underlying the antipruritic effect. Our results indicate that drugs targeting α2 and α3GABAA receptors are well-suited to alleviate itch, including non-histaminergic chronic itch for which currently no approved treatment exists.Chronic itch affects about 10% of the general population, however current treatments are largely ineffective. Here, the authors show that targeting of inhibitory α2 and α3GABAA receptors reduces itch in mice and in a canine model, suggesting this a potentially useful therapeutic approach.

Prevention of an increase in cortical ligand binding to AMPA receptors may represent a novel mechanism of endogenous brain protection by G-CSF after ischemic stroke

PURPOSEnUsing G-CSF deficient mice we recently demonstrated neuroprotective properties of endogenous G-CSF after ischemic stroke. The present follow-up study was designed to check, whether specific alterations in ligand binding densities of excitatory glutamate or inhibitory GABAA receptors may participate in this effect.nnnMETHODSnThree groups of female mice were subjected to 45 minutes of MCAO: wildtype, G-CSF deficient and G-CSF deficient mice substituted with G-CSF. Infarct volumes were determined after 24 hours and quantitative in vitro receptor autoradiography was performed using [3H]MK-801, [3H]AMPA and [3H]muscimol for labeling of NMDA, AMPA and GABAA receptors, respectively. Ligand binding densities were analyzed in regions in the ischemic core, peri-infarct areas and corresponding contralateral regions.nnnRESULTSnInfarct volumes did not significantly differ between the experimental groups. Ligand binding densities of NMDA and GABAA receptors were widely in the same range. However, AMPA receptor binding densities in G-CSF deficient mice were substantially enhanced compared to wildtype mice. G-CSF substitution in mice lacking G-CSF largely reversed this effect.nnnCONCLUSIONSnAlthough infarct volumes did not differ 24 hours after ischemia the increase of AMPA receptor binding densities in G-CSF deficient mice may explain the bigger infarcts previously observed at later time-points with the same stroke model.