Peripheral Administration Research Articles

Peripheral intravenous Ceftriaxone administration can cause upper extremity deep vein thrombosis in patients with heart failure

Peripheral intravenous Ceftriaxone administration can cause upper extremity deep vein thrombosis in patients with heart failure

Lipopolysaccharide inhibits hypothalamic Agouti-related protein gene expression via activating mechanistic target of rapamycin signaling in chicks

Lipopolysaccharide (LPS) induces profound anorexia in birds. However, the neuronal regulatory network underlying LPS-provoked anorexia is unclear. To determine whether any cross talk occurs among hypothalamic mechanistic target of rapamycin (mTOR) and LPS in the regulation of appetite, we performed an intracerebroventricular injection of rapamycin (an mTOR inhibitor) on LPS-treated chicks. The results indicate that peripheral administrations of LPS decreased the agouti-related protein (AgRP) mRNA level, but increased the phosphorylated mTOR and nuclear factor-кB (NF-кB) protein level. Blocking mTOR significantly attenuated LPS-induced anorexia, AgRP suppression, and p-NF-кB increase. Thus, the results suggest that LPS causes anorexia via the mTOR-AgRP signaling pathway, and mTOR signaling is also associated with the regulation of LPS in p-NF-кB.

Oxytocin attenuates schizophrenia-like reduced sensorimotor gating in outbred and inbred rats in line with strain differences in CD38 gene expression

Prepulse inhibition (PPI) of the startle response is a measure of sensorimotor gating that is impaired in many clinical conditions, including schizophrenia. The inbred Roman high-avoidance (RHA) rats, compared to their low-avoidance (RLA) counterparts, show distinct schizophrenia-like phenotypes, such as spontaneous deficits in PPI accompanied by decreased medial prefrontal cortex (mPFC) activity and volume. Schizophrenia-like deficits are usually attenuated by antipsychotic drugs, but these drugs often produce severe side effects. In order to reduce these side effects, the neuropeptide oxytocin has been proposed as an alternative natural antipsychotic for schizophrenia. Here, we examined the effects of peripheral oxytocin administration (saline, 0.04, and 0.2 mg/kg) on PPI in the RHA vs. RLA rats, as well as in the outbred heterogeneous stock (HS) rats. Our results showed that oxytocin increased PPI in the HS rats and attenuated PPI deficits in the RHA rats, but it did not significantly affect PPI in the RLAs. To explore whether these divergent effects were associated with differences in oxytocinergic mechanisms, we analyzed gene expression of the oxytocin receptor (OXTR) and the regulator of oxytocin release (CD38) in the mPFC of the Roman rats. Consistent with the differential oxytocin effects on PPI (RHA > RLA), constitutive CD38 expression was reduced in the RHA rats compared to the RLAs, while oxytocin administration increased OXTR expression in both strains. Overall, the present work reveals that oxytocin administration shows antipsychotic-like effects on PPI in outbred and inbred rats, and it suggests that these effects may be related to basal differences in oxytocin-mediated mechanisms in the mPFC.

L-Cell Expression of Melanocortin-4-Receptor Is Marginal in Most of the Small Intestine in Mice and Humans and Direct Stimulation of Small Intestinal Melanocortin-4-Receptors in Mice and Rats Does Not Affect GLP-1 Secretion.

The molecular sensors underlying nutrient-stimulated GLP-1 secretion are currently being investigated. Peripheral administration of melanocortin-4 receptor (MC4R) agonists have been reported to increase GLP-1 plasma concentrations in mice and humans but it is unknown whether this effect results from a direct effect on the GLP-1 secreting L-cells in the intestine, from other effects in the intestine or from extra-intestinal effects. We investigated L-cell expression of MC4R in mouse and human L-cells by reanalyzing publicly available RNA sequencing databases (mouse and human) and by RT-qPCR (mouse), and assessed whether administration of MC4R agonists to a physiologically relevant gut model, isolated perfused mouse and rat small intestine, would stimulate GLP-1 secretion or potentiate glucose-stimulated secretion. L-cell MC4R expression was low in mouse duodenum and hardly detectable in the ileum and MC4R expression was hardly detectable in human L-cells. In isolated perfused mouse and rat intestine, neither intra-luminal nor intra-arterial administration of NDP-alpha-MSH, a potent MC4R agonist, had any effect on GLP-1 secretion (P ≥0.98, n = 5–6) from the upper or lower-half of the small intestine in mice or in the lower half in rats. Furthermore, HS014—an often used MC4R antagonist, which we found to be a partial agonist—did not affect the glucose-induced GLP-1 response in the rat, P = 0.62, n = 6). Studies on transfected COS7-cells confirmed bioactivity of the used compounds and that concentrations employed were well within in the effective range. Our combined data therefore suggest that MC4R-activated GLP-1 secretion in rodents either exclusively occurs in the colon or involves extra-intestinal signaling.

Peripheral Administration of NMU Promotes White Adipose Tissue Beiging and Improves Glucose Tolerance.

Purpose Targeting white adipose tissue (WAT) beiging has been proposed as an effective way to increase thermogenesis and improve glucose metabolism. Neuromedin U (NMU) is a neuropeptide that could increase energy expenditure, while its effects on WAT beiging and glucose homeostasis remain to be investigated. Methods Male C57BL/6 mice were fed with high fat diet (HFD) to induce obesity and hyperglycemia and then treated with chronic subcutaneous injection of NMU. Body weight and food intake were recorded daily. After 14 days of injection, intraperitoneal glucose tolerance tests and 18F-fluorodeoxyglucose micro-positron emission tomography/computed tomography (18F-FDG micro-PET/CT) scans were conducted. Subcutaneous WAT (sWAT) and interscapular brown adipose tissue were collected for the evaluation of adipocyte size, expression of uncoupling protein 1 (Ucp1), and other thermogenic-related genes. Stromal vascular fraction of subcutaneous WAT was extracted for the measurement of type 2 innate lymphocytes (ILC2s) proportions. Results Glucose tolerance was markedly improved by peripherally administered NMU. Micro-PET/CT suggested that NMU promoted WAT beiging, which was further confirmed by haematoxylin and eosin (H&E) staining and immunohistochemistry. In diet-induced-obese (DIO) mice, NMU activated thermogenic-related genes in WAT. In addition, NMU stimulated ILC2s in the stromal vascular fraction of WAT. Conclusion Taken together, our study indicates that peripheral administration of NMU is a potential therapeutic strategy for the promotion of WAT beiging and the improvement of impaired glucose tolerance.

Oxytocin administration in neonates shapes hippocampal circuitry and restores social behavior in a mouse model of autism

Oxytocin is an important regulator of the social brain. In some animal models of autism, notably in Magel2tm1.1Mus-deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms, focusing on social memory performance. Using in situ hybridization (RNAscope), we have established that Magel2 and oxytocin receptor are co-expressed in the dentate gyrus and CA2/CA3 hippocampal regions involved in the circuitry underlying social memory. Then, we have shown that Magel2tm1.1Mus-deficient mice, evaluated in a three-chamber test, present a deficit in social memory. Next, in hippocampus, we conducted neuroanatomical and functional studies using immunostaining, oxytocin-binding experiments, ex vivo electrophysiological recordings, calcium imaging and biochemical studies. We demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin receptors and of somatostatin interneurons in both DG and CA2/CA3 regions. We also revealed a delay in the GABAergic development sequence in Magel2tm1.1Mus-deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring hippocampal alterations and social memory at adulthood. Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.

Intravenous administration of LPS activates the kynurenine pathway in healthy male human subjects: a prospective placebo-controlled cross-over trial

BackgroundAdministration of lipopolysaccharide (LPS) from Gram-negative bacteria, also known as the human endotoxemia model, is a standardized and safe model of human inflammation. Experimental studies have revealed that peripheral administration of LPS leads to induction of the kynurenine pathway followed by depressive-like behavior and cognitive dysfunction in animals. The aim of the present study is to investigate how acute intravenous LPS administration affects the kynurenine pathway in healthy male human subjects.MethodsThe present study is a prospective, single-blinded, randomized, placebo-controlled cross-over study to investigate the effects of intravenously administered LPS (Escherichia coli O113, 2 ng/kg) on tryptophan and kynurenine metabolites over 48 h and their association with interleukin-6 (IL-6) and C-reactive protein (CRP). The study included 10 healthy, non-smoking men (18–40 years) free from medication. Statistical differences in tryptophan and kynurenine metabolites as well as associations with IL-6 and CRP in LPS and placebo treated subjects were assessed with linear mixed-effects models.ResultsSystemic injection of LPS was associated with significantly lower concentrations of plasma tryptophan and kynurenine after 4 h, as well as higher concentrations of quinolinic acid (QUIN) after 48 h compared to the placebo injection. No differences were found in kynurenic acid (KYNA) or picolinic acid plasma concentrations between LPS or placebo treatment. The KYNA/kynurenine ratio peaked at 6 h post LPS injection while QUIN/kynurenine maintained significantly higher from 3 h post LPS injection until 24 h. The kynurenine/tryptophan ratio was higher at 24 h and 48 h post LPS treatment. Finally, we report an association between the kynurenine/tryptophan ratio and CRP.ConclusionsOur findings strongly support the concept that an inflammatory challenge with LPS induces the kynurenine pathway in humans, activating both the neurotoxic (QUIN) and neuroprotective (KYNA) branch of the kynurenine pathway.Trial registrationThis study is based on a study registered at ClinicalTrials.gov, NCT03392701. Registered 21 December 2017.

O-BN002. Intravenous administration of poly(lactide-co-glycolide) nanoparticle encapsulating brain-derived neurotrophic factor promotes neuroprotection in an ischemic stroke model in the rat

Introduction . Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) are biodegradable and biocompatible drug carriers and competent at delivering neuroprotective agents to the brain following peripheral administration. Brain-derived neurotrophic factor (BDNF) could provide neuroprotection in brain insult particularly ischaemic injury. We tested the neuroprotective effect of PLGA nanoparticle-bound BDNF (NPBDNF) on a permanent middle cerebral artery occlusion (pMCAO) model of ischemic rats. Methods . Sprague-Dawley rats were divided into 4 groups of 7 rats each. Group 1 was subjected to sham operation, group 2, 3, and 4 were subjected to pMCAO. Four hours after pMCAO, group 3 and 4 were intravenously (IV) treated with BDNF and NPBDNF, respectively at 4 hours post-ischaemic induction. Functional outcome was assessed at 2 and 24 hours after pMCAO, using the modified Neurologic Severity Score (mNSS) and grid walking. Following functional assessments, rats were euthanized by terminal cardiac puncture, whereby blood was taken to assess for neurobiomarker level, the neuron spesific enolase (NSE). The brain was evaluated to measure the infarct volume. Results . The NPBDNF treated group showed significant functional improvement in mNSS, demonstrating a mild injury evidenced by a decreased in mNSS score by 2.0 and 2.1 times, respectively, when compared with pMCAO and BDNF treated groups. Grid walking test showed significant improvement by error reduction in contralateral foot slips in rats treated with NPBDNF as compared with BDNF alone by 2.00 fold. The infarct volume in rats treated with NPBDNF was significantly smaller by 1.91 fold compared to the pMCAO group and 1.95 fold compared to the BDNF treated group. These results were further corroborated by the NSE level estimates. Conclusion . NPBDNF exhibit a significant neuroprotective effect in the pMCAO model of ischemia in rats following intravenous administration during the critical period of neuroprotective window.

Incidence and challenges of helicopter emergency medical service (HEMS) rescue missions with helicopter hoist operations: analysis of 11,228 daytime and nighttime missions in Switzerland

ObjectiveWe aimed to investigate the medical characteristics of helicopter hoist operations (HHO) in HEMS missions.MethodsWe designed a retrospective study evaluating all HHO and other human external cargo (HEC) missions performed by Swiss Air-Rescue (Rega) between January 1, 2010, and December 31, 2019.ResultsDuring the study period, 9,963 (88.7 %) HEMS missions with HHO and HEC were conducted during the day, and 1,265 (11.3 %) at night. Of the victims with time-critical injuries (NACA ≥ 4), 21.1 % (n = 400) reached the hospital within 60 min during the day, and 9.1 % (n = 18) at night. Nighttime missions, a trauma diagnosis, intubation on-site, and NACA Score ≥ 4 were independently and highly significantly associated with longer mission times (p < 0.001). The greatest proportion of patients who needed hoist or HEC operations in the course of the HEMS mission during the daytime sustained moderate injuries (NACA 3, n = 3,731, 37.5 %) while practicing recreational activities (n = 5,492, 55.1 %). In daytime HHO missions, the most common medical interventions performed were insertion of a peripheral intravenous access (n = 3,857, 38.7 %) and administration of analgesia (n = 3,121, 31.3 %).ConclusionsNearly 20 % of patients who needed to be evacuated by a hoist were severely injured, and complex and lifesaving medical interventions were necessary before the HHO procedure. Therefore, only adequately trained and experienced medical crew members should accompany HHO missions.

Infusion of hESC derived Immunity-and-matrix regulatory cells improves cognitive ability in early-stage AD mice.

ObjectivesIn this study, we administered immunity‐and‐matrix regulatory cells (IMRCs) via tail vein (IV) and intracerebroventricular (ICV) injection to 3‐month‐old 5×FAD transgenic mice to assess the effects of IMRC transplantation on the behaviour and pathology of early‐stage Alzheimer's disease (AD).Materials and methodsClinical‐grade human embryonic stem cell (hESC)‐derived IMRCs were produced under good manufacturing practice (GMP) conditions. Three‐month‐old 5×FAD mice were administered IMRCs via IV and ICV injection. After 3 months, the mice were subjected to behavioural tests and electrophysiological analysis to evaluate their cognitive function, memory ability and synaptic plasticity. The effect of IMRCs on amyloid‐beta (Aβ)‐related pathology was detected by thioflavin‐S staining and Western blot. Quantitative real‐time PCR, ELISA and immunostaining were used to confirm that IMRCs inhibit neuroinflammation. RNA‐seq analysis was performed to measure changes in gene expression and perform a pathway analysis in response to IMRC treatment.ResultsIMRC administration via tail vein injection significantly ameliorated cognitive deficits in early‐stage AD (5×FAD) mice. However, no significant change was observed in the characteristic pathology of AD in the ICV group. Plaque analysis revealed that IMRCs did not influence either plaque deposition or BACE1 expression. In addition, IMRCs inhibited inflammatory responses and reduced microglial activation in vivo.ConclusionsWe have shown that peripheral administration of IMRCs can ameliorate AD pathology and associated cognitive deficits.

Risk factor identification and predictive models for central line requirements for patients on vasopressors.

Vasopressors are ubiquitous in intensive care units. While central venous catheters are the preferred route of infusion, recent evidence suggests peripheral administration may be safe for short, single-agent courses. Here, we identify risk factors and develop a predictive model for patient central venous catheter requirement using the Medical Information Mart for Intensive Care, a single-centre dataset of patients admitted to an intensive care unit between 2008 and 2019. Using prior literature, a composite endpoint of prolonged single-agent courses (>24 hours) or multi-agent courses of any duration was used to identify likely central venous catheter requirement. From a cohort of 69,619 intensive care unit stays, there were 17,053 vasopressor courses involving one or more vasopressors that met study inclusion criteria. In total, 3807 (22.3%) vasopressor courses involved a single vasopressor for less than six hours, 7952 (46.6%) courses for less than 24 hours and 5757 (33.8%) involved multiple vasopressors of any duration. Of these, 3047 (80.0%) less than six-hour and 6423 (80.8%) less than 24-hour single vasopressor courses used a central venous catheter. Logistic regression models identified associations between the composite endpoint and intubation (odds ratio (OR) 2.36, 95% confidence intervals (CI) 2.16 to 2.58), cardiac diagnosis (OR 0.72, CI 0.65 to 0.80), renal impairment (OR 1.61, CI 1.50 to 1.74), older age (OR 1.002, Cl 1.000 to 1.005) and vital signs in the hour before initiation (heart rate, OR 1.006, CI 1.003 to 1.009; oxygen saturation, OR 0.996, CI 0.993 to 0.999). A logistic regression model predicting the composite endpoint had an area under the receiver operating characteristic curve (standard deviation) of 0.747 (0.013) and an accuracy of 0.691 (0.012). This retrospective study reveals a high prevalence of short vasopressor courses in intensive care unit settings, a majority of which were administered using central venous catheters. We identify several important risk factors that may help guide clinicians deciding between peripheral and central venous catheter administration, and present a predictive model that may inform future prospective trials.

Subcutaneous infusion of neurosecretory protein GL promotes fat accumulation in mice

We recently identified a novel small secretory protein, neurosecretory protein GL (NPGL), in the vertebrate hypothalamus. We revealed that NPGL is involved in energy homeostasis using intracerebroventricular infusion in rodents. However, the effect of NPGL through peripheral administration remains to be elucidated and may be important for therapeutic use. In this study, we performed subcutaneous infusion of NPGL in mice for 12 days and found that it accelerated fat accumulation in white adipose tissue (WAT) without increasing in body mass gain and food intake. The mass of the testis, liver, kidney, heart, and gastrocnemius muscle remained unchanged. Analysis of mRNA expression by quantitative reverse transcription-polymerase chain reaction showed that proopiomelanocortin was suppressed in the hypothalamus by the infusion of NPGL. We observed a decreasing tendency in serum triglyceride levels due to NPGL, while serum glucose, insulin, leptin, and free fatty acids levels were unchanged. These results suggest that the peripheral administration of NPGL induces fat accumulation in WAT via the hypothalamus.

Role of TRPM8 in switching between fever and hypothermia in adult mice during endotoxin-induced inflammation

Transient receptor potential melastatin 8 (TRPM8) functions in the sensing of noxious and innocuous colds; however, its significance in pathogen-induced thermoregulation remains unclear. In the present study, we investigated the role of TRPM8 in the regulation of endotoxin-induced body temperature control. The peripheral administration of low-dose lipopolysaccharide (LPS) at 50 ​μg/kg generated fever in wild-type (WT) mice, whereas it caused hypothermia in TRPM8 knockout (KO) animals. LPS-induced sickness responses such as decrease in body weight, and food and water intake were not different between WT and TRPM8 KO mice. TRPM8 KO mice exhibited more severe hypothermia and lower locomotor activity following the peripheral administration of high-dose LPS at 5 ​mg/kg compared with WT ones. An intracerebroventricular (i.c.v.) injection of either LPS at 3.6 ​μg/kg or interleukin-1β at 400 ​ng/kg elicited hypothermia in TRPM8 KO mice, in contrast to fever in WT animals. The peripheral administration of zymosan at 3 ​mg/kg also induced hypothermia in contrast to fever in WT mice. An i.c.v. injection of prostaglandin E2 at 16 or 160 ​nmol/kg induced normal fever in both WT and TRPM8 KO mice. Infrared thermography showed significant decline of the interscapular skin temperature that estimates temperature of the brown adipose tissue, regardless of no alteration of its temperature in WT animals. Fos immunohistochemistry showed stronger Fos activation of hypothalamic thermoregulation-associated nuclei in TRPM8 KO mice compared with WT animals following the peripheral administration of low-dose LPS. Therefore, the present study indicates that TRPM8 is necessary for switching between fever and hypothermia during endotoxin-induced inflammation.

Safety and Efficacy of 23.4% Sodium Chloride Administered via Peripheral Venous Access for the Treatment of Cerebral Herniation and Intracranial Pressure Elevation.

Sodium chloride (NaCl) 23.4% solution has been shown to reduce intracranial pressure (ICP) and reverse transtentorial herniation. A limitation of 23.4% NaCl is its high osmolarity (8008mOsm/l) and the concern for tissue injury or necrosis following extravasation when administered via peripheral venous access. The use of this agent is therefore often limited to central venous or intraosseous routes of administration. Our objective was to evaluate the safety and efficacy of administration of 23.4% NaCl via peripheral venous access compared with administration via central venous access. We reviewed pharmacy records to identify all administrations of 23.4% NaCl at our institution between December 2017 and February 2020. Medical records were then reviewed to identify complications, such as extravasation, soft tissue injury or necrosis, hypotension (mean arterial pressure less than 65mm Hg), pulmonary edema, hemolysis, and osmotic demyelination. We also compared the change in physiological variables, such as ICP, mean arterial pressure, cerebral perfusion pressure, and heart rate, as well as laboratory values, such as sodium, chloride, bicarbonate, creatinine, and hemoglobin, following administration of 23.4% NaCl via the peripheral and central venous routes. We identified 299 administrations of 23.4% NaCl (242 central and 57 peripheral) in 141 patients during the study period. There was no documented occurrence of soft tissue injury or necrosis in any patient. One patient developed hypotension following central administration. Among the 38 patients with ICP monitoring at the time of drug administration, there was no significant difference in median ICP reduction (- 13mm Hg [central] vs. - 24mm Hg [peripheral], p = 0.21) or cerebral perfusion pressure augmentation (16mm Hg [central] vs. 15mm Hg [peripheral], p = 0.87) based on route of administration. Peripheral venous administration of 23.4% NaCl is safe and achieves a reduction in ICP equivalent to that achieved by administration via central venous access.

Liver-Directed Gene Therapy for Hemophilia B with Immune Stealth Lentiviral Vectors

Gene therapy is rapidly gaining renewed interest and is emerging as a realistic treatment option for several genetic and acquired diseases. Underlying this success is the development of improved gene transfer vectors. Among them, lentiviral vectors (LV) are emerging as versatile vehicles of relatively large capacity for stable transgene integration in the genome of target cells. Over the past years, we developed LV that achieve stable transgene expression in the liver and establish correction of hemophilia, in mouse and dog models of the disease, upon systemic administration. These LV are designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We previously evaluated portal vein administration of LV expressing canine factor IX (FIX) in 3 adult hemophilia B dogs and reported stable multiyear reconstitution of FIX activity up to 1 % of normal. We have more recently treated 4 dogs as pups (2-4 months of age) by peripheral vein administration of escalating doses of LV (5 to 10e9 T.U./kg) expressing a hyper-functional canine FIX and achieved reconstitution of FIX activity up to 32% of normal at the highest dose tested. We then modified the vector envelope by changing the protein composition of the producer cell plasma membrane through genome editing to reduce immunogenicity of LV particles. First, we performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all class-I major histocompatibility complexes (MHC-I) to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. These MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with autologous monocytes exposed to LV, from several healthy donors, suggesting that conventional MHC-bearing LV may trigger allogeneic immune responses. Secondly, we generated LV with increasedlevels of CD47, a phagocytosis inhibitor, on the vector surface (CD47high LV), which show substantially decreased uptake by human macrophages in vitro . In order to evaluate the role of CD47 in LV biodistribution upon in vivo administration we took advantage of the non-obese diabetic (NOD) mouse model whose SIRPα (the CD47 receptor) is known to have high affinity for the human CD47. In this setting, CD47 proved to be a key player in reducing phagocytosis of LV, decreasing the inflammatory cytokine response following their administration, and increasing hepatocyte transduction and FIX output. We have now administered MHC-free or MHC-free/CD47high LV to 6 non-human primates (NHP, 3 for each LV version). We chose Macaca nemestrina as recipient, because of the lower restriction to HIV infection than other NHP species. Administration of 7.5e9 T.U./kg LV via peripheral vein was well tolerated, without significant elevation of serum aminotransferases or body temperature and only caused a transient self-limiting leukopenia. Remarkably, human-specific FIX activity reached up to 300% of normal and was nearly 3-fold higher in the CD47high-LV treated animals, showing a much more favorable LV dose-response than observed in mice and dogs. Upon necropsy, we measured vector copies in liver, spleen and major organs of treated animals and found between 0.5 and 1.5 LV copies in the liver accounting for 80-90% of all the retrieved LV copies, showing selective targeting and efficient gene transfer to the liver by LV in NHP. Overall, our studies support the efficacy and safety of these immune-stealth LV in NHP and position them to address some of the outstanding challenges in liver-directed gene therapy for hemophilia and conceivably other diseases. DisclosuresLiu:Bioverativ: Employment, Other: Shareholder. Peters:Bioverativ: Employment, Other: Shareholder.

Peripheral administration of SOD1 aggregates does not transmit pathogenic aggregation to the CNS of SOD1 transgenic mice

The deposition of aggregated proteins is a common neuropathological denominator for neurodegenerative disorders. Experimental evidence suggests that disease propagation involves prion-like mechanisms that cause the spreading of template-directed aggregation of specific disease-associated proteins. In transgenic (Tg) mouse models of superoxide dismutase-1 (SOD1)-linked amyotrophic lateral sclerosis (ALS), inoculation of minute amounts of human SOD1 (hSOD1) aggregates into the spinal cord or peripheral nerves induces premature ALS-like disease and template-directed hSOD1 aggregation that spreads along the neuroaxis. This infectious nature of spreading pathogenic aggregates might have implications for the safety of laboratory and medical staff, recipients of donated blood or tissue, or possibly close relatives and caregivers. Here we investigate whether transmission of ALS-like disease is unique to the spinal cord and peripheral nerve inoculations or if hSOD1 aggregation might spread from the periphery into the central nervous system (CNS). We inoculated hSOD1 aggregate seeds into the peritoneal cavity, hindlimb skeletal muscle or spinal cord of adult Tg mice expressing mutant hSOD1. Although we used up to 8000 times higher dose—compared to the lowest dose transmitting disease in spinal cord inoculations—the peripheral inoculations did not transmit seeded aggregation to the CNS or premature ALS-like disease in hSOD1 Tg mice. Nor was any hSOD1 aggregation detected in the liver, kidney, skeletal muscle or sciatic nerve. To explore potential reasons for the lack of disease transmission, we examined the stability of hSOD1 aggregates and found them to be highly vulnerable to both proteases and detergent. Our findings suggest that exposed individuals and personnel handling samples from ALS patients are at low risk of any potential transmission of seeded hSOD1 aggregation.

Role of GABAAR in the Transition From Acute to Chronic Pain and the Analgesic Effect of Electroacupuncture on Hyperalgesic Priming Model Rats.

Chronic pain is a costly health problem that impairs health-related quality of life when not effectively treated. Regulating the transition from acute to chronic pain is a new therapeutic strategy for chronic pain that presents a major clinical challenge. The underlying mechanisms of pain transition are not entirely understood, and strategies for preventing this transition are lacking. Here, a hyperalgesic priming model was used to study the potential mechanism by which γ-aminobutyric acid receptor type A (GABAAR) in the dorsal root ganglion (DRG) contributes to pain transition. Furthermore, electroacupuncture (EA), a modern method of acupuncture, was administered to regulate pain transition, and the mechanism underlying EA’s regulatory effect was investigated. Hyperalgesic priming was induced by intraplanar injection of carrageenan (Car)/prostaglandin E2 (PGE2). The decrease in mechanical withdrawal threshold (MWT) induced by PGE2 returned to baseline 4 h after injection in NS + PGE2 group, and still persisted 24 h after injection in Car + PGE2 group. Lower expression of GABAAR in the lumbar DRG was observed in the model rats. Furthermore, activating or blocking GABAAR could reversed the long-lasting hyperalgesia induced by Car/PGE2 injection or produced a persistent hyperalgesia. In addition, GABAAR may be involved in Protein Kinase C epsilon (PKCε) activation in the DRG, a mark molecular of pain transition. EA considerably increased the mechanical pain thresholds of hyperalgesic priming model mammals in both the acute and chronic phases. Furthermore, EA upregulated the expression of GABAAR and inhibited the activation of PKCε in the DRG. In addition, peripheral administration of picrotoxin blocked the analgesic effect of EA on the model rats and abolished the regulatory effect of EA on PKCε activation. These findings suggested that GABAAR plays a key role in both the transition from acute to chronic pain and the analgesic effect of EA on hyperalgesic priming.

Re-exposure to alarmin HMGB1 and cytokine IL-1 beta induces differential innate immune response generation in mouse brain

Innate immune memory, a crucial mechanism of epigenetically mediated myeloid cell plasticity, alters subsequent immune responses majorly by two types of immunological imprinting, training, and tolerance. Recent pioneer studies provided proof-of-principle for generation of both types of innate immune memory in brain macrophage, microglial cells. This novel study was designed to investigate whether the pattern of immune response generation, induced by peripheral administration of recombinant alarmin HMGB1, alone and in combination with other recombinant cytokines, is affected by prior exposure. The experimental outcomes revealed that full length recombinant HMGB1 exposure for seven consecutive days exhibit inflammatory response as evidenced by enhanced expression of inflammatory biomarkers and neurodegeneration. In contrary, combined doses of HMGB1 and IL-1β, for three and seven consecutive days, exhibited lower inflammatory state compared to its alone HMGB1 counterpart. The immune tolerance state was evident by microglial polarization towards non-reactive M2 state, lower astrocyte activation, epigenetic reprogramming, and decreased neurodegeneration. This is the first demonstration that HMGB1 and IL-1β priming can differentially affect inflammation in the brain when a host is confronted with a second, third stimulus or so on. The findings were further validated by suppressing major regulators of epigenetic reprogramming, by intranasal delivery of specific siRNAs targeting those regulators. These results may provide new evidence for the involvement of recombinant endogenous cytokine induced generation of innate immune tolerance within microglial cells and indicated the possible potential role in mediating cognitive and behavioural alterations during inflammatory diseases.

Peripheral cortisol administration blunts reward arousal but heightens anxiety-like arousal in marmosets

AimsExcess hypothalamic-pituitary-adrenal (HPA) axis activation is common in people with major depression and generalised anxiety disorder. We sought to determine whether higher circulating levels of the glucocorticoid cortisol are causally related to the expression of anhedonia-like and anxiety-like behaviours in marmosets.MethodFour marmosets (two male, two female) took part in the study. Cortisol and saline control injections were administered intramuscularly and salivary cortisol samples were taken before and after injections to determine if circulating cortisol levels changed from pre- to post-injection. To measure anhedonia-like behaviours, we trained marmosets on an appetitive Pavlovian conditioning paradigm, where animals learn to associate two anticipatory auditory cues (conditioned stimulus + or conditioned sitmulus -, CS+ or CS-) with the presence or absence of food reward (unconditioned stimulus + or unconditioned stimulus -, US+ or US-). Using cardiovascular telemetry probes and video cameras, we recorded animals' cardiovascular and behavioural arousal in freely moving conditions, comparing the injection of saline control versus 5mg/kg, 10mg/kg or 20mg/kg intramuscular cortisol. To measure anxiety-like behaviours, we used a human intruder (HI) paradigm, where marmosets are confronted with an unfamiliar human in their home cage. We recorded their behaviour on video cameras after saline control or 20mg/kg intramuscular cortisol. We used an exploratory-factor analysis (EFA) to determine how marmosets' behaviours towards the intruder loaded onto an 'anxiety-like' score. We then compared these scores under saline control versus cortisol conditions. Significance was set at p &lt; 0.05.ResultUnlike saline control, we found that subcutaneous injections of 20 mg/kg cortisol successfully elevated peripheral cortisol concentrations to levels equivalent to peak circadian concentrations (p = 0.023). In the appetitive setting, 5 mg/kg, 10 mg/kg and 20 mg/kg cortisol injections blunted anticipatory (CS+ induced) increases in behavioural arousal (p = 0.004) but did not alter anticipatory cardiovascular arousal. Consummatory behavioural and cardiovascular arousal also remained intact. In the HI test, 20 mg/kg cortisol injections moderately increased anxiety towards the intruder as measured by an increase in marmosets' EFA-derived anxiety-like scores (p = 0.035).ConclusionIn marmosets, elevated peripheral cortisol levels are causally related to the behavioural features of blunted reward anticipation together with elevated anxiety-like behaviours characteristic of mood and anxiety disorders. Future work will characterise the neuroimaging changes induced by elevated peripheral cortisol levels and identify the regions of the prefrontal cortex contributing to HPA axis regulation and dysregulation.

EVALUATION OF ANTINOCICEPTIVE EFFECT OF THE KETOROLAC-TOPIRAMATE COMBINATION IN THE RAT FORMALIN TEST

Combination therapy approaches to manage acute and chronic pain are commonly used. To characterize the interaction between ketorolac and topiramate in the formalin test, female Wistar rats (200-300 g) were submitted to 1% formalin test. Antinociceptive effect was determined by the administration of ketorolac (3, 10, 30 and 100 mg/kg), topiramate (25, 50, 100 and 200 mg/kg) and their combination by oral route; or ketorolac (25, 50, 100 and 200 µg/paw), topiramate (10, 30, 100 and 300 µg/paw) and their combination by local peripheral route. Isobolographic analysis was used in a fixed dose combination (0.5:0.5) to analyze the nature of the interaction of the combination based on the ED50 of ketorolac (62.8±21.9 mg/kg) and topiramate (24.3±8.5 mg/kg) by oral administration; or the ED30 of ketorolac (67.0±7.97 µg/paw) and topiramate (391.3±51.7 µg/paw) by local peripheral route. Combination of these two drugs significantly reduced the number of flinches in second phase of the test. Theoretical ED50 of the oral combination (ED50T) was 43.5±11.7 mg/kg. Experimentally, the ED50 of the combination (ED50E) had a significantly lower value: 16.7±4.0 mg/kg; indicating the presence of supra additive effects (interaction index was 0.38). For local peripheral route, ED30T was 229.21±64.99 µg/kg. Experimentally, the ED30E had a significantly lower value: 62.00±15.72 µg/paw; indicating synergistic effects (interaction index was 0.27). Results show that oral and local peripheral administration of the combination can interact synergistically to reduce inflammatory pain in the rat formalin test.