Layer-specific changes of KCC2 and NKCC1 in the mouse dentate gyrus after entorhinal denervation

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl− concentration and cell volume of neurons and/or glia. The Cl− extruder KCC2 is expressed at higher levels than the Cl− transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl− concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis

IκB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-κB in the central nervous system

The IκB kinase complex induces nuclear factor kappa B activation and has recently been recognized as a key player of autoimmunity in the central nervous system. Notably, IκB kinase/nuclear factor kappa B signalling regulates peripheral myelin formation by Schwann cells, however, its role in myelin formation in the central nervous system during health and disease is largely unknown. Surprisingly, we found that brain-specific IκB kinase 2 expression is dispensable for proper myelin assembly and repair in the central nervous system, but instead plays a fundamental role for the loss of myelin in the cuprizone model. During toxic demyelination, inhibition of nuclear factor kappa B activation by conditional ablation of IκB kinase 2 resulted in strong preservation of central nervous system myelin, reduced expression of proinflammatory mediators and a significantly attenuated glial response. Importantly, IκB kinase 2 depletion in astrocytes, but not in oligodendrocytes, was sufficient to protect mice from myelin loss. Our results reveal a crucial role of glial cell-specific IκB kinase 2/nuclear factor kappa B signalling for oligodendrocyte damage during toxic demyelination. Thus, therapies targeting IκB kinase 2 function in non-neuronal cells may represent a promising strategy for the treatment of distinct demyelinating central nervous system disease

倫理創成研究の動向 : シュレーダー＝フレチェットの『リスクと合理性』から

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Upregulation of APP, ADAM10 and ADAM17 in the denervated mouse dentate gyrus

The disintegrin and metalloproteinases ADAM10 and ADAM17 are regarded as the most important α-secretases involved in the physiological processing of amyloid precursor protein (APP) in brain. Since it has been suggested that processing of APP by α-secretases could be involved in the reorganization of the brain following injury, we studied mRNA expression of the two α-secretases Adam10 and Adam17, the ß-secretase Bace1, and the App-gene family (App, Aplp1, Aplp2) in the dentate gyrus of the mouse following entorhinal denervation. Using laser microdissection, tissue was harvested from the outer molecular layer and the granule cell layer of the denervated dentate gyrus. Expression levels of candidate genes were assessed using Affymetrix GeneChip Mouse Gene 1.0 ST arrays and reverse transcription-quantitative PCR, revealing an upregulation of Adam10 mRNA and Adam17 mRNA in the denervated outer molecular layer and an upregulation of Adam10 mRNA and App mRNA in the dentate granule cell layer. Immunolabeling for ADAM10 or ADAM17 in combination with markers for astro- and microglia revealed an increased labeling of ADAM10 and ADAM17 in the denervated outer molecular layer that was associated with reactive astrocytes but not with microglia. Collectively, these data show that denervation affects the expression level of APP and its two most important α-secretases. This suggests that APP-processing could be shifted towards the non-amyloidogenic pathway in denervated areas of the brain and, thus, towards the formation of neuroprotective APP cleavage products, such as APPsα

Precise measurement of gene expression changes in mouse brain areas denervated by injury

Abstract Quantitative PCR (qPCR) is a widely used method to study gene expression changes following brain injury. The accuracy of this method depends on the tissue harvested, the time course analyzed and, in particular on the choice of appropriate internal controls, i.e., reference genes (RGs). In the present study we have developed and validated an algorithm for the accurate normalization of qPCR data using laser microdissected tissue from the mouse dentate gyrus after entorhinal denervation at 0, 1, 3, 7, 14 and 28 days postlesion. The expression stabilities of ten candidate RGs were evaluated in the denervated granule cell layer (gcl) and outer molecular layer (oml) of the dentate gyrus. Advanced software algorithms demonstrated differences in stability for single RGs in the two layers at several time points postlesion. In comparison, a normalization index of several stable RGs covered the entire post-lesional time course and showed high stability. Using these RGs, we validated our findings and quantified glial fibrillary acidic protein (Gfap) mRNA and allograft inflammatory factor 1 (Aif1/Iba1) mRNA in the denervated oml. We compared the use of single RGs for normalization with the normalization index and found that single RGs yield variable results. In contrast, the normalization index gave stable results. In sum, our study shows that qPCR can yield precise, reliable, and reproducible datasets even under such complex conditions as brain injury or denervation, provided appropriate RGs for the model are used. The algorithm reported here can easily be adapted and transferred to any other brain injury model

Regulation of the spatial code for BDNF mRNA isoforms in the rat hippocampus following pilocarpine-treatment: A systematic analysis using laser microdissection and quantitative real-time PCR

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and plasticity and is one of those genes that generate multiple mRNAs with different alternatively spliced 5'UTRs. The functional significance of many BDNF transcripts, each producing the same protein, is emerging. On the basis of the analysis of the four most abundant brain BDNF transcripts, we recently proposed the "spatial code hypothesis of BDNF splice variants" according to which the BDNF transcripts, through their differential subcellular localization in soma or dendrites, represent a mechanism to synthesize the protein at distinct locations and produce local effects. In this study, using laser microdissection of hippocampal laminae and reverse transcription-quantitative real-time PCR (RT-qPCR), we analyzed all known BDNF mRNA variants at resting conditions or following 3 h pilocarpine-induced status epilepticus. In untreated rats, we found dendritic enrichment of BDNF transcripts encoding exons 6 and 7 in CA1; exons 1, 6, and 9a in CA3; and exons 5, 6, 7, and 8 in DG. Considering the low abundance of the other transcripts, exon 6 was the main transcript in dendrites under resting conditions. Pilocarpine treatment induced an increase of BDNF transcripts encoding exons 4 and 6 in all dendritic laminae and, additionally, of exon 2 in CA1 stratum radiatum and exons 2, 3, 9a in DG molecular layer while the other transcripts were decreased in dendrites, suggesting restriction to the soma. These results support the hypothesis of a spatial code to differentially regulate BDNF in the somatic or dendritic compartment under conditions of pilocarpine-induced status epilepticus and, furthermore, highlight the existence of subfield-specific differences

Maturation-dependent differences in the re-innervation of the denervated dentate gyrus by sprouting associational and commissural mossy cell axons in organotypic tissue cultures of entorhinal cortex and hippocampus

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2- hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro, which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations