Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Immune and Neural Response to Acute Social Stress in Adolescent Humans and Rodents.

Studies in adults have linked stress-related activation of the immune system to the manifestation of psychiatric conditions. Using a translational design, this study aimed to examine the impact of social stress on immune activity in adolescents and on neuronal activity in a preclinical mouse model. Participants were 31 adolescents (ages 12-19), including 25 with mood and anxiety symptoms. Whole-blood samples were collected before and after the Trier Social Stress Test (TSST), a stress-inducing public speaking task, then cultured for 6 hours in the presence and absence of the inflammatory endotoxin lipopolysaccharide (LPS). Effects of TSST and LPS on 41 immune biomarkers were examined using repeated-measures analysis of variance. Separately, juvenile (8-week-old) male mice were non-stressed or exposed to reminder social defeat then intraperitoneally injected with saline or LPS (n = 6/group). Brains were perfused and collected for immunohistochemistry and confocal microscopy at 0, 1, 6, and 24 hours post-injection. Activity was determined by the density of cFos-positive neurons in the paraventricular hypothalamus, paraventricular thalamus, and basolateral amygdala, regions known to show sustained activation to immunological challenge. Analyses in the adolescent study indicated a strong effect of LPS but no effects of TSST or TSST×LPS interaction on immune biomarkers. Similarly, reminder social defeat did not induce sustained neuronal activity changes comparable to LPS immunological challenge in juvenile mice. Our convergent findings across species suggest that the acute immune response to stress documented in adults is not present in youth. Thus, aging and chronicity effects may play an important role in the inflammatory response to acute psychosocial stress.

Ventilation Management in a Patient with Ventilation-Perfusion Mismatch in the Early Phase of Lung Injury and during the Recovery.

Chest trauma is one of the most serious and difficult injuries, with various complications that can lead to ventilation-perfusion (V/Q) mismatch and systemic hypoxia. We are presenting a case of a 53-year-old male with no chronic therapy who was admitted to the Intensive Care Unit due to severe respiratory failure after chest trauma. He developed a right-sided pneumothorax, and then a thoracic drain was placed. On admission, the patient was hemodynamically unstable and tachypneic. He was intubated and mechanically ventilated, febrile (38.9 °C) and unconscious. A lung CT showed massive non-ventilated areas, predominantly in the right lung, guiding repeated therapeutic and diagnostic bronchoalveolar lavages. He was ventilated with PEEP of 10 cmH2O with a FiO2 of 0.6-0.8. Empirical broad-spectrum antimicrobial therapy was immediately initiated. Both high FiO2 and moderate PEEP were maintained and adjusted according to the current blood gas values and oxygen saturation. He was weaned from mechanical ventilation, and non-invasive oxygenation was continued. After Stenotrophomonas maltophilia was identified and treated with sulfamethoxazole/trimethoprim, a regression of lung infiltrates was observed. In conclusion, both ventilatory and antibiotic therapy were needed to improve the oxygenation and outcome of the patient with S. maltophilia pneumonia and V/Q mismatch.

Long-Term Outcomes of Breast Cancer Patients Receiving Levobupivacaine Wound Infiltration or Diclofenac for Postoperative Pain Relief.

Breast cancer is the most common malignant disease in women. Preclinical studies have confirmed that the local anesthetic levobupivacaine has a cytotoxic effect on breast cancer cells. We examined whether postoperative wound infiltration with levobupivacaine influences survival in 120 patients who were operated on for breast cancer and underwent quadrantectomy or mastectomy with axillary lymph node dissection. Groups with continuous levobupivacaine wound infiltration, bolus wound infiltration, and diclofenac analgesia were compared. Long-term outcomes examined were quality of life, shoulder disability, and hand grip strength (HGS) after one year and survival after 5 and 10 years. Groups that had infiltration analgesia had better shoulder function compared to diclofenac after one year. The levobupivacaine PCA group had the best-preserved HGS after 1 year (P = 0.022). The most significant predictor of the 5-year outcome was HGS (P = 0.03). Survival at 10 years was 85%, 92%, and 77% in the diclofenac, levobupivacaine bolus, and levobupivacaine PCA groups (ns. P = 0.36). The extent of the disease at the time of surgery is the most important predictor of long-term survival (P = 0.03). A larger prospective clinical study could better confirm the effect of levobupivacaine wound infiltration on outcomes after breast cancer surgery observed in this pilot study-trial number NCT05829707.

miRNA-132/212 Deficiency Disrupts Selective Corticosterone Modulation of Dorsal vs. Ventral Hippocampal Metaplasticity.

Cortisol is a potent human steroid hormone that plays key roles in the central nervous system, influencing processes such as brain neuronal synaptic plasticity and regulating the expression of emotional and behavioral responses. The relevance of cortisol stands out in the disease, as its dysregulation is associated with debilitating conditions such as Alzheimer's Disease, chronic stress, anxiety and depression. Among other brain regions, cortisol importantly influences the function of the hippocampus, a structure central for memory and emotional information processing. The mechanisms fine-tuning the different synaptic responses of the hippocampus to steroid hormone signaling remain, however, poorly understood. Using ex vivo electrophysiology and wild type (WT) and miR-132/miR-212 microRNAs knockout (miRNA-132/212-/-) mice, we examined the effects of corticosterone (the rodent's equivalent to cortisol in humans) on the synaptic properties of the dorsal and ventral hippocampus. In WT mice, corticosterone predominantly inhibited metaplasticity in the dorsal WT hippocampi, whereas it significantly dysregulated both synaptic transmission and metaplasticity at dorsal and ventral regions of miR-132/212-/- hippocampi. Western blotting further revealed significantly augmented levels of endogenous CREB and a significant CREB reduction in response to corticosterone only in miR-132/212-/- hippocampi. Sirt1 levels were also endogenously enhanced in the miR-132/212-/- hippocampi but unaltered by corticosterone, whereas the levels of phospo-MSK1 were only reduced by corticosterone in WT, not in miR-132/212-/- hippocampi. In behavioral studies using the elevated plus maze, miRNA-132/212-/- mice further showed reduced anxiety-like behavior. These observations propose miRNA-132/212 as potential region-selective regulators of the effects of steroid hormones on hippocampal functions, thus likely fine-tuning hippocampus-dependent memory and emotional processing.

Advances in Analgosedation and Periprocedural Care for Gastrointestinal Endoscopy.

The number and complexity of endoscopic gastrointestinal diagnostic and therapeutic procedures is globally increasing. Procedural analgosedation during gastrointestinal endoscopic procedures has become the gold standard of gastrointestinal endoscopies. Patient satisfaction and safety are important for the quality of the technique. Currently there are no uniform sedation guidelines and protocols for specific gastrointestinal endoscopic procedures, and there are several challenges surrounding the choice of an appropriate analgosedation technique. These include categories of patients, choice of drug, appropriate monitoring, and medical staff providing the service. The ideal analgosedation technique should enable the satisfaction of the patient, their maximum safety and, at the same time, cost-effectiveness. Although propofol is the gold standard and the most used general anesthetic for endoscopies, its use is not without risks such as pain at the injection site, respiratory depression, and hypotension. New studies are looking for alternatives to propofol, and drugs like remimazolam and ciprofol are in the focus of researchers' interest. New monitoring techniques are also associated with them. The optimal technique of analgosedation should provide good analgesia and sedation, fast recovery, comfort for the endoscopist, patients' safety, and will have financial benefits. The future will show whether these new drugs have succeeded in these goals.

Gastrointestinal Motility Disorders Correlate with Intracranial Bleeding, Opioid Use, and Brainstem Edema in Neurosurgical Patients.

BACKGROUND: Gastrointestinal (GI) motility disorders may be directly associated with the intensity of acute brain injury, edema of the brainstem, and opioid use in neurosurgical patients. METHODS: In this retrospective study, patient demographic characteristics, computed tomography (CT) scans, the occurrence of gastroparesis, constipation, and opioid use were registered during the intensive care unit (ICU) stay and correlated with days of mechanical ventilation, length of ICU stay, and survival. Gastroparesis was defined as residual gastric volume > 250 mL per day, and constipation was defined as the absence of stool for 3 days or more during the ICU stay. RESULTS: Of 207 neurosurgical patients screened, 69 adult patients who spent more than 4 days in the ICU were included in the study. Gastroparesis was observed in 48 (69.6%) patients, constipation was observed in 67 (97.1%) patients, and stress ulcers were observed in 4 (5.8%) patients. Patients with brainstem edema (n = 57, 82.6%) had the first stool evacuation later compared with patients with no edema (8 [interquartile range (IQR) 5.25-9.75] vs. 3.5 [IQR 2.25-4] days; P < 0.001). In the logistic regression analysis, factors that were associated with GI dysmotility were central nervous system (CNS) bleeding (odds ratio [OR] 5.1, 95% confidence interval [CI] 1.26-20.8, P = 0.02), opioid use > 19.3 morphine equivalents (ME) per day (OR 5.37, 95% CI 1.1-27.1, P = 0.04), and brainstem edema (OR 4.9, 95% CI 1.1-21.6, P = 0.04). A receiver operating characteristic curve analysis confirmed that the cutoff value of > 6.78 ME per day was a good predictor determining GI dysmotility, with 89.5% sensitivity and 72.7% specificity (95% CI 0.67-0.88, area under the curve 0.784, Youden index 0.62, P = 0.001). Poor survival correlated with lower Glasgow Coma Score values (ρ = - 520, P < 0.001), CNS bleeding (ρ = 0.393, P < 0.001), associated cardiac diseases (ρ = 0.279, P < 0.001), and cardiorespiratory arrest on admission (ρ = 0.315, P < 0.001), but not with GI dysmotility (ρ = 0.175, P = 0.402). CONCLUSIONS: Significant correlation was registered between brainstem edema, gastrointestinal dysmotility, and opioids. CNS bleeding was the most important single factor influencing GI dysmotility. Further studies with opioid and nonopioid sedation may distinguish the influence of acute brain lesions versus drugs on GI dysmotility.

CHARACTERISTICS AND TREATMENT OUTCOMES OF UROLOGIC PATIENTS ADMITTED TO THE INTENSIVE CARE UNIT, OSIJEK UNIVERSITY HOSPITAL CENTER.

The postoperative care unit at the Department of Urology has significantly improved treatment of patients undergoing surgical procedures and reduced admission of urologic patients to the Intensive Care Unit (ICU). We examined the characteristics of urologic patients, time on mechanical ventilation, most common complications, and mortality in the period from January 2017 to March 2022. A total of 84 admissions to ICU were recorded, accounting for 1.5% of all patients having undergone surgical, therapeutic or diagnostic interventions under general or regional anesthesia at the Department of Urology. The most common reasons for admission to ICU were respiratory failure (79 patients), hemodynamic instability, and bleeding. The median time on mechanical ventilation was 9.7 [2.4-58.2] hours in urology patients vs. 6 [3-14.7] hours in the rest of surgical ICU patients (p=0.058). Hypertension and renal failure were more common in urologic than in the rest of surgical ICU patients (p<0.05). The overall mortality of urologic patients was lower than in the rest of surgical ICU patients (10.7% vs. 18.99%, p=0.08) but the difference did not reach statistical significance. Independently of the lower mortality, improvements in the outcome of urologic patients admitted to the ICU are feasible. Early identification of patients at risk of infections, postoperative respiratory failure, cardiovascular incidents, and bleeding may further reduce mortality and improve outcomes.

Management of Anesthesia and Perioperative Procedures in a Child with Glucose-6-Phosphate Dehydrogenase Deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that helps red blood cells work properly; it participates in the production of antioxidants and helps to defend cells against oxidative damage. With all this in mind, patients with G6PD deficiency may be very sensitive and vulnerable to different oxidative stressors, because they can cause some serious medical conditions of which hemolytic anemia is common in adults and severe jaundice in newborns. The most common triggers of hemolysis in G6PD deficiency are infections, medications, metabolic conditions such as diabetic ketoacidosis, hypothermia, and a very important item-surgical stress. During the operative period, the anesthetic goal is to reduce stress and monitor if the hemolysis occurs, and of course, treat it if it occurs. In our case, the combination of sevoflurane inhalation anesthesia with the addition of sufentanil proved to be safe and effective in the management of a child with G6PD deficiency.

FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder.

Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

Stress-induced changes of the cholinergic circuitry promote retrieval-based generalization of aversive memories.

Generalization, the process of applying knowledge acquired in one context to other contexts, often drives the expression of similar behaviors in related situations. At the cellular level, generalization is thought to depend on the activity of overlapping neurons that represent shared features between contexts (general representations). Using contextual fear conditioning in mice, we demonstrate that generalization can also occur in response to stress and result from reactivation of specific, rather than general context representations. We found that generalization emerges during memory retrieval, along with stress-induced abnormalities of septohippocampal oscillatory activity and acetylcholine release, which are typically found in negative affective states. In hippocampal neurons that represent aversive memories and drive generalization, cholinergic septohippocampal afferents contributed to a unique reactivation pattern of cFos, Npas4, and repressor element-1 silencing transcription factor (REST). Together, these findings suggest that generalization can be triggered by perceptually dissimilar but valence-congruent memories of specific aversive experiences. Through promoting the reactivation of such memories and their interference with ongoing behavior, abnormal cholinergic signaling could underlie maladaptive cognitive and behavioral generalization linked to negative affective states.

Protocol for assessing the role of hippocampal perineuronal nets in aversive memories.

Perineuronal nets (PNNs) are emerging as critical regulators of memory-related neuronal processes. However, their exact contribution depends on type of memory, consolidation stage, or brain region, and remains to be fully investigated. We describe here a protocol to evaluate the importance of PNNs in the dorsal hippocampus in different stages of aversive memories using a mouse model. The protocol provides detailed instructions for surgical implantation of hippocampal cannulas, drug infusion, contextual fear conditioning procedures, and immunohistochemistry for PNN visualization. For complete details on the use and execution of this protocol, please refer to Jovasevic et al. (2021).

miRNA-132/212 Gene-Deletion Aggravates the Effect of Oxygen-Glucose Deprivation on Synaptic Functions in the Female Mouse Hippocampus.

Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved short-length/noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA-132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia.

STAT3 in the dorsal raphe gates behavioural reactivity and regulates gene networks associated with psychopathology.

The signal transducer and activator of transcription 3 (STAT3) signalling pathway is activated through phosphorylation by Janus kinases in response to a diverse set of immunogenic and non-immunogenic triggers. Several distinct lines of evidence propose an intricate involvement of STAT3 in neural function relevant to behaviour in health and disease. However, in part due to the pleiotropic effects resulting from its DNA binding activity and the consequent regulation of expression of a variety of genes with context-dependent cellular consequences, the precise nature of STAT3 involvement in the neural mechanisms underlying psychopathology remains incompletely understood. Here, we focused on the midbrain serotonergic system, a central hub for the regulation of emotions, to examine the relevance of STAT3 signalling for emotional behaviour in mice by selectively knocking down raphe STAT3 expression using germline genetic (STAT3 KO) and viral-mediated approaches. Mice lacking serotonergic STAT3 presented with reduced negative behavioural reactivity and a blunted response to the sensitising effects of amphetamine, alongside alterations in midbrain neuronal firing activity of serotonergic neurons and transcriptional control of gene networks relevant for neuropsychiatric disorders. Viral knockdown of dorsal raphe (DR) STAT3 phenocopied the behavioural alterations of STAT3 KO mice, excluding a developmentally determined effect and suggesting that disruption of STAT3 signalling in the DR of adult mice is sufficient for the manifestation of behavioural traits relevant to psychopathology. Collectively, these results suggest DR STAT3 as a molecular gate for the control of behavioural reactivity, constituting a mechanistic link between the upstream activators of STAT3, serotonergic neurotransmission and psychopathology.

Stress-related memories disrupt sociability and associated patterning of hippocampal activity: a role of hilar oxytocin receptor-positive interneurons.

In susceptible individuals, memories of stressful experiences can give rise to debilitating socio-affective symptoms. This occurs even when the ability to retrieve such memories is limited, as seen in patients suffering from traumatic amnesia. We therefore hypothesized that the encoding, rather than retrieval, mechanisms of stress-related memories underlie their impact on social and emotional behavior. To test this hypothesis, we used combinations of stress-enhanced and state-dependent fear conditioning, which engage different encoding mechanisms for the formation of stress-related memories. We found that the encoding of stress-enhanced state-dependent memories robustly and sex specifically impairs sociability in male mice and disrupts the asymmetry of dentate gyrus (DG)/CA3 activity accompanying social interactions. These deficits were restored by chemogenetic inactivation of oxytocin receptor-positive interneurons localized in the hilus (Oxtr-HI), and by inactivation of dorsohippocampal efferents to the caudal lateral septum. Together, our data suggest that disrupted patterning of dorsohippocampal DG/CA3 activity underlies stress-induced sociability deficits, and that Oxtr-HI can be a cellular target for improving these deficits.

Severe hydroxymethylbilane synthase deficiency causes depression-like behavior and mitochondrial dysfunction in a mouse model of homozygous dominant acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant inborn error of heme biosynthesis due to a pathogenic mutation in the Hmbs gene, resulting in half-normal activity of hydroxymethylbilane synthase. Factors that induce hepatic heme biosynthesis induce episodic attacks in heterozygous patients. The clinical presentation of acute attacks involves the signature neurovisceral pain and may include psychiatric symptoms. Here we used a knock-in mouse line that is biallelic for the Hmbs c.500G > A (p.R167Q) mutation with ~ 5% of normal hydroxymethylbilane synthase activity to unravel the consequences of severe HMBS deficiency on affective behavior and brain physiology. Hmbs knock-in mice (KI mice) model the rare homozygous dominant form of AIP and were used as tool to elucidate the hitherto unknown pathophysiology of the behavioral manifestations of the disease and its neural underpinnings. Extensive behavioral analyses revealed a selective depression-like phenotype in Hmbs KI mice; transcriptomic and immunohistochemical analyses demonstrated aberrant myelination. The uncovered compromised mitochondrial function in the hippocampus of knock-in mice and its ensuing neurogenic and neuroplastic deficits lead us to propose a mechanistic role for disrupted mitochondrial energy production in the pathogenesis of the behavioral consequences of severe HMBS deficiency and its neuropathological sequelae in the brain.