Intraperitoneal Injection Of Saline Research Articles

T-cell responses in colorectal peritoneal metastases are recapitulated in a humanized immune system mouse model

BackgroundThe occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC.MethodsWe studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF).ResultsThe human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses.ConclusionIn conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.

T-cell responses in colorectal peritoneal metastases are recapitulated in a humanized immune system mouse model

BackgroundThe occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC.MethodsWe studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF).ResultsThe human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses.ConclusionIn conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.

Augmentation of functional recovery via ROCK/PI3K/AKT pathway by Fasudil Hydrochloride in a rat sciatic nerve transection model

BackgroundsThe functional recovery after peripheral nerve injury remains unsatisfactory. This study aims to perform a comprehensive evaluation of the efficacy of Fasudil Hydrochloride at treating the sciatic nerve transection injury in rats and the mechanism involved. Materials and methodsIn animal experiments, 75 Sprague Dawley rats that underwent transection and repair of the right sciatic nerve were divided into a control, Fasudil, and Fas + LY group, receiving daily intraperitoneal injection of saline, Fasudil Hydrochloride (10 mg/kg), and Fasudil Hydrochloride plus LY294002 (5 mg/kg), respectively. At day 3 after surgery, the expression of ROCK2, p-PI3K, and p-AKT in L4-5 DRG and the lumbosacral enlargement was determined using Western blotting. At day 7 and 14, axon density in the distal stump was evaluated with immunostaining using the anti-Neurofilament-200 antibody. At day 30, retrograde tracing by injecting Fluoro-gold in the distal stump was performed. Three months after surgery, remyelination was analyzed with immostaining using the anti-MPZ antibody and the transmission electron microscope; Moreover, Motion-Evoked Potential, and recovery of sensorimotor functions was evaluated with a neuromonitor, Footprint, Hot Plate and Von Frey Filaments, respectively. Moveover, the Gastrocnemius muscles were weighed, and then underwent H＆E staining, and staining of the neuromuscular junction using α-Bungarotoxin to evaluate the extent of atrophy and degeneration of the endplates in the Gastrocnemius. In vitro, spinal motor neurons (SMNs) and dorsal root ganglia (DRG) were cultured to examine the impact of Fasudil Hydrochloride and LY294002 on the axon outgrowth. ResultsThree days after injury, the expression of ROCK2 increased significantly (P＜0.01), and Fasudil application significantly increased the expression of p-PI3K and p-AKT in L4-6 DRG and the lumbosacral enlargement (P < 0.05). At day 7 and 14 after surgery, a higher axon density could be observed in the Fasudil group(P < 0.05). At day 30 after surgery, a larger number of motor and sensory neurons absorbing Fluoro-gold could be observed in the Fasudil group (P < 0.01) Three months after surgery, a greater thickness of myelin sheath could be observed in the Fasudil group (P < 0.05). The electrophysiological test showed that a larger amplitude of motion-evoked potential could be triggered in the Fasudil group (P < 0.01). Behavioral tests showed that a higher sciatic function index and a lower threshold for reacting to heat and mechanical stimuli could be measured in the Fasudil group. (P < 0.01). The wet weight ratio of the Gastrocnemius muscles and the area of the cross section of its myofibrils were greater in the Fasudil group (P < 0.01), which also demonstrated a higer ratio of axon-endplate connection and a larger size of endplates (P < 0.05). And there were no significant differences for the abovementioned parameters between the control and Fas + LY groups (P＞0.05). In vitro studies showed that Fasudil could significantly promote axon growth in DRG and SMNs, and increase the expression of p-PI3K and p-AKT, which could be abolished by LY294002 (P < 0.05). ConclusionsFasudil can augment axon regeneration and remyelination, and functional recovery after sciatic nerve injury by activating the PI3K/AKT pathway. The translational potential of this articleThe translation potential of this article is that we report for the first time that Fasudil Hydrochloride has a remarkable efficacy at improving axon regeneration and remyelination following a transection injury of the right sciatic nerve in rats through the ROCK/PI3K/AKT pathway, which has a translational potential to be used clinically to treat peripheral nerve injury.

Psilocybin decreases neural responsiveness and increases functional connectivity while preserving pure-tone frequency selectivity in mouse auditory cortex.

Psilocybin is a serotonergic psychedelic believed to have therapeutic potential for neuropsychiatric conditions. Despite well-documented prevalence of perceptual alterations, hallucinations, and synesthesia associated with psychedelic experiences, little is known about how psilocybin affects sensory cortex or alters the activity of neurons in awake animals. To investigate, we conducted two-photon imaging experiments in auditory cortex of awake mice and collected video of free-roaming mouse behavior, both at baseline and during psilocybin treatment. In comparison with pre-dose neural activity, a 2 mg/kg ip dose of psilocybin initially increased the amplitude of neural responses to sound. Thirty minutes post-dose, behavioral activity and neural response amplitudes decreased, yet functional connectivity increased. In contrast, control mice given intraperitoneal saline injections showed no significant changes in either neural or behavioral activity across conditions. Notably, neuronal stimulus selectivity remained stable during psilocybin treatment, for both tonotopic cortical maps and single-cell pure-tone frequency tuning curves. Our results mirror similar findings regarding the effects of serotonergic psychedelics in visual cortex and suggest that psilocybin modulates the balance of intrinsic versus stimulus-driven influences on neural activity in auditory cortex.NEW & NOTEWORTHY Recent studies have shown promising therapeutic potential for psychedelics in treating neuropsychiatric conditions. Musical experience during psilocybin-assisted therapy is predictive of treatment outcome, yet little is known about how psilocybin affects auditory processing. Here, we conducted two-photon imaging experiments in auditory cortex of awake mice that received a dose of psilocybin. Our results suggest that psilocybin modulates the roles of intrinsic neural activity versus stimulus-driven influences on auditory perception.

#539 The active ingredient in the Nuphar lutea plant DTBN, ameliorates kidney damage and inflammation in a mouse model of chronic kidney disease (CKD)

Abstract Background and Aims Complications associated with Chronic Kidney Disease (CKD), such as disease progression and anemia, are closely linked to heightened inflammation mediated by the innate immune response. This inflammation can be mitigated through the use of anti-inflammatory agents. 6,6′-dihydroxythiobinupharidine (DTBN), an active compound derived from the aquatic plant Nuphar lutea, demonstrates notable anti-inflammatory properties, primarily by inhibiting NF-κB and LPS-induced cytokine activation. DTBN has shown efficacy in priming neutrophils, enhancing phagocytosis, regulating reactive oxygen species (ROS) production, and modulating NET formation. This study aimed to assess the impact of DTBN on CKD in mice. Method Male C57BL/6J mice, aged 8 weeks, were subjected to an adenine diet known to induce CKD through crystal deposition and tubulointerstitial inflammation. Both control and CKD-induced mice received intraperitoneal injections of DTBN (25 μg QOD) or saline and were euthanized after 8 weeks for evaluation. Results CKD mice treated with DTBN exhibited a significant decrease in serum urea and creatinine levels compared to untreated CKD mice (reduction by 1.4-fold and 1.3-fold, respectively). Histological analysis of kidney tissues revealed a notable reduction in F4/80 positive macrophage infiltration (3.4-fold decrease) and damaged renal areas (three-fold decrease), along with diminished kidney TGF-β levels in DTBN-treated CKD mice. Moreover, the p65 subunit of NF-κB showed a three-fold reduction in CKD mice treated with DTBN compared to untreated CKD mice. Indices of kidney inflammation (IL-1β, IL-6, and P-STAT3) were notably decreased in CKD mice treated with DTBN (reduction by 1.4-fold, 1.6-fold, and 2.9-fold, respectively) compared to untreated CKD mice. Notably, DTBN treatment in control mice did not induce apparent tissue damage; however, it led to reduced weight gain and mild hypoalbuminemia without proteinuria. Conclusion DTBN demonstrated significant improvements in renal function and inflammation indices in CKD mice. Hence, DTBN and substances similar to DTBN could be considered as supplementary treatments for CKD patients.

CCR5 antagonist maraviroc alleviates doxorubicin-induced neuroinflammation and neurobehavioral deficiency by regulating NF-κB/NLRP3 signaling in a breast cancer mouse model

The chemotherapeutic agent Doxorubicin (DOX) is known to cause chemotherapy-induced cognitive impairment (CICI). Maraviroc, a potent C–C chemokine receptor 5 (CCR5) antagonist, shows neuroprotective properties, while its role in CICI remains unclear. This study determined the therapeutic potential of maraviroc on CICI. Adult C57BL/6J mice with implanted breast cancer cells received four weekly intraperitoneal injections of saline (Control group), 5 mg/kg DOX (DOX group), 10 mg/kg maraviroc (MVC group), or 5 mg/kg DOX with 10 mg/kg maraviroc (DOX + MVC group). The Morris Water Maze (MWM) was used for neurobehavioural test. Western blot analysis and immunofluorescence were used to evaluate the expressions of inflammatory markers, apoptosis-related proteins, and synaptic-related proteins. The volume and weight of tumor were also evaluated after treatments. DOX treatment significantly increased chemokines (CCL3, CCL4) and inflammatory cytokines (IL-1β, TNF-α) in tumor-bearing mice hippocampus. While maraviroc administration reduced hippocampal proinflammatory factors compared to the DOX group. Furthermore, it also lowered apoptosis markers, restored synaptic proteins levels, and inhibited the NF-κB/NLRP3 pathway. Accordingly, maraviroc treatment significantly improved DOX-induced neurobehavioural impairments as evidenced by an increased number of platform crossings and percentage of target quadrant time in the MWM test. Additionally, when combined with DOX, maraviroc had additional inhibitory effects on tumor growth. These findings suggest that maraviroc can mitigate DOX-induced CICI by suppressing elevated proinflammatory chemokines and cytokines through the NF-κB/NLRP3 pathway, potentially offering an anti-tumor benefit. This research presents a promising therapeutic approach for DOX-induced CICI, enhancing the safety and efficacy of cancer treatments.

Pain experience reduces social avoidance to others in pain: a c-Fos-based functional connectivity network study in mice.

Pain experience increases individuals' perception and contagion of others' pain, but whether pain experience affects individuals' affiliative or antagonistic responses to others' pain is largely unknown. Additionally, the neural mechanisms underlying how pain experience modulates individuals' responses to others' pain remain unclear. In this study, we explored the effects of pain experience on individuals' responses to others' pain and the underlying neural mechanisms. By comparing locomotion, social, exploration, stereotyped, and anxiety-like behaviors of mice without any pain experience (naïve observers) and mice with a similar pain experience (experienced observers) when they observed the pain-free demonstrator with intraperitoneal injection of normal saline and the painful demonstrator with intraperitoneal injection of acetic acid, we found that pain experience of the observers led to decreased social avoidance to the painful demonstrator. Through whole-brain c-Fos quantification, we discovered that pain experience altered neuronal activity and enhanced functional connectivity in the mouse brain. The analysis of complex network and graph theory exhibited that functional connectivity networks and activated hub regions were altered by pain experience. Together, these findings reveal that neuronal activity and functional connectivity networks are involved in the modulation of individuals' responses to others' pain by pain experience.

Hepatic steatosis induced by nicotine plus Coca-Cola™ is prevented by nicotinamide riboside (NR).

Cigarettes containing nicotine (Nic) are a risk factor for the development of cardiovascular and metabolic diseases. We reported that Nic delivered via injections or e-cigarette vapor led to hepatic steatosis in mice fed with a high-fat diet. High-fructose corn syrup (HFCS) is the main sweetener in sugar-sweetened beverages (SSBs) in the US. Increased consumption of SSBs with HFCS is associated with increased risks of non-alcoholic fatty liver disease (NAFLD). Nicotinamide riboside (NR) increases mitochondrial nicotinamide adenine dinucleotide (NAD+) and protects mice against hepatic steatosis. This study evaluated if Nic plus Coca-Cola™ (Coke) with HFCS can cause hepatic steatosis and that can be protected by NR. C57BL/6J mice received twice daily intraperitoneal (IP) injections of Nic or saline and were given Coke (HFCS), or Coke with sugar, and NR supplementation for 10 weeks. Our results show that Nic+Coke caused increased caloric intake and induced hepatic steatosis, and the addition of NR prevented these changes. Western blot analysis showed lipogenesis markers were activated (increased cleavage of the sterol regulatory element-binding protein 1 [SREBP1c] and reduction of phospho-Acetyl-CoA Carboxylase [p-ACC]) in the Nic+Coke compared to the Sal+Water group. The hepatic detrimental effects of Nic+Coke were mediated by decreased NAD+ signaling, increased oxidative stress, and mitochondrial damage. NR reduced oxidative stress and prevented mitochondrial damage by restoring protein levels of Sirtuin1 (Sirt1) and peroxisome proliferator-activated receptor coactivator 1-alpha (PGC1) signaling. We conclude that Nic+Coke has an additive effect on producing hepatic steatosis, and NR is protective. This study suggests concern for the development of NAFLD in subjects who consume nicotine and drink SSBs with HFCS.

L-citrulline Mitigates Systemic Inflammation and Associated Pulmonary Vascular Remodeling in a Neonatal Rat Model of Postnatal Undernutrition

Background &amp; Aim: Preterm infants with postnatal growth restriction and undernutrition are at increased risk of developing bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), metabolic syndrome, and sepsis. Preclinical and clinical studies report increased systemic inflammation, and lung and pancreatic histopathology in growth-restricted neonates. Safe therapies to mitigate these adverse outcomes associated with postnatal growth restriction and undernutrition in preterm infants are needed. L-citrulline is a non-essential amino acid, involved in the urea and nitric oxide cycles, that exhibits anti-inflammatory properties. This study aims to investigate whether L-citrulline may decrease systemic inflammation and improve histopathology in the lung, heart, and pancreas in a neonatal model of postnatal undernutrition. Methods: Neonatal Sprague Dawley rat pups were divided into 2 groups, normal nutrition (litter size n=12) and undernutrition (litter size n=18) by altering the litter size of the nursing dam. Body weight was measured daily and both groups received daily intraperitoneal injections of saline or L-citrulline (2.5g/kg) for 21 days. On postnatal day 21, heart, lung, and pancreatic tissue were collected for histology and mRNA analyses. Lung and pancreatic tissue were processed for qPCR to detect mRNA expression of pro-inflammatory mediators (MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α). Right ventricular hypertrophy was calculated using Fulton’s index (weight of the right ventricle divided by the combined weight of the left ventricle and the intraventricular septum). Lung sections were stained with Movat Pentachrome and examined for the presence of immune cells and medial wall thickening of pulmonary arteries. In a separate group of animals, bronchoalveolar lavage fluid (BALF) and blood obtained by cardiac puncture were cytocentrifuged and stained with hematoxylin &amp; eosin for immune cell counts. Results: Undernourished rats had a lower body weight than normally nourished rats at postnatal day 21 (n=11-17; p&lt;0.0001). Undernourished animals had an increased number of immune cells in BALF and lung tissue and an elevated neutrophil count in the blood which were all decreased with concurrent L-citrulline treatment (n=3-4; p&lt;0.001). L-citrulline treatment also prevented the increased mRNA expression of pro-inflammatory mediators in the lungs [MCP-1/CCL2, TNF-α, and IL-1β] (n=8; p&lt;0.05) and pancreas [MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α] (n=3; p&lt;0.05) of undernourished animals. These animals had a greater pulmonary artery medial wall area and Fulton’s index when compared to normally nourished animals treated with saline or L-citrulline (n=3-4; p&lt;0.001). Concurrent L-citrulline treatment in undernourished animals prevented pulmonary artery vascular remodeling and heart tissue histopathology such that the medial wall area and Fulton’s index did not differ from that of animals in control groups (n=4; p&lt;0.01). Conclusion: L-citrulline effectively mitigates the adverse effects of postnatal undernutrition, reducing lung, pancreatic, and systemic inflammation and histopathology in the lung and heart associated with PH. As a safe, well-tolerated, and inexpensive treatment, L-citrulline presents promising therapeutic potential that could prevent short and long-term morbidities associated with growth restriction and undernutrition in preterm infants. This work was funded by the Women's Auxiliary of The Hospital for Sick Children. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Systemic Co-Administration of Low-Dose Oxytocin and Glucagon-Like Peptide 1 Additively Decreases Food Intake and Body Weight

Introduction: GLP-1 receptor agonists are the number one drug prescribed for the treatment of obesity and type 2 diabetes. These drugs are not, however, without side effects, and in an effort to maximize therapeutic effect while minimizing adverse effects, gut hormone co-agonists received considerable attention as new drug targets in the fight against obesity. Numerous previous reports identified the neuropeptide oxytocin (OXT) as a promising anti-obesity drug. The aims of this study were to evaluate OXT as a possible co-agonist for GLP-1 and examine the effects of its co-administration on food intake (FI) and body weight (BW) in mice. Methods: FI and c-Fos levels were measured in the feeding centers of the brain in response to an intraperitoneal injection of saline, OXT, GLP-1, or OXT/GLP-1. The action potential frequency and cytosolic Ca2+ ([Ca2+]i) in response to OXT, GLP-1, or OXT/GLP-1 were measured in ex vivo paraventricular nucleus (PVN) neuronal cultures. Finally, FI and BW changes were compared in diet-induced obese mice treated with saline, OXT, GLP-1, or OXT/GLP-1 for 13 days. Results: Single injection of OXT/GLP-1 additively decreased FI and increased c-Fos expression specifically in the PVN and supraoptic nucleus. Seventy percent of GLP-1 receptor-positive neurons in the PVN also expressed OXT receptors, and OXT/GLP-1 co-administration dramatically increased firing and [Ca2+]i in the PVN OXT neurons. The chronic OXT/GLP-1 co-administration decreased BW without changing FI. Conclusion: Chronic OXT/GLP-1 co-administration decreases BW, possibly via the activation of PVN OXT neurons. OXT might be a promising candidate as an incretin co-agonist in obesity treatment.

The role of Cannabinoid receptors in visceral pain sensation of rat: an interventional study

Visceral pain, originating from internal organs, represents a challenging aspect of pain management due to its intricate mechanisms and often debilitating nature. Understanding the underlying pathways involved in visceral pain perception is crucial for developing effective therapeutic strategies. The current study aimed to delve into recent advancements in the understanding of cannabinergic modulation of visceral pain perception, focusing on findings from interventional studies utilizing animal models, particularly rats. A total of 30 male rats aged 3 months, with an average weight of 220 g were randomly divided into 3 groups. The groups contained the control group which received intraperitoneal injection of normal saline, the second group received an intraperitoneal injection of anandamide (2 mg/kg), and the third group received an intraperitoneal injection of tramadol (20 mg/kg). The pain in all groups assessed by acetic acid test. The data obtained from the intraperitoneal injection of anandamide to the rats of the experimental group showed a significant decrease in the amount of perceived visceral pain compared to control group. In addition, the results showed that tramadol injection significantly decreased visceral pain in experimental group 2 compared to the control group. In conclusion, the current study provides an evidence for the involvement of cannabinoid receptors in the modulation of visceral pain sensation in rats.

507 Neuroprotective Effects of a Steroid Receptor Coactivator Stimulator in a Murine Model of Acute Ischemic Stroke with Reperfusion

INTRODUCTION: Stroke is the leading cause of adult disability and the fifth leading cause of mortality in the United States. Despite the study of many neuroprotective agents for acute ischemic stroke, none has been shown to be effective in large randomized clinical trials. Steroid receptor coactivator (SRC) is a promising class of agents since they are involved in cellular proliferation and regeneration, immune modulation, angiogenesis, antioxidant protection and are expressed in the brain. METHODS: Twenty c57bl/6 mice were randomly assigned to control or treatment groups (n = 10 mice per group). All mice underwent middle cerebral artery occlusion for 90 minutes followed by reperfusion via the intraluminal filament method. Occlusion was confirmed by laser doppler flowmetry. Intraperitoneal injections of saline (control) or 10-1 (treatment) were given 30 minutes after reperfusion. Each animal was tested using a modified Bederson’s neurological deficit scale (mNDS) and euthanized at the conclusion of the 24-hour survival period. Brain slices were stained with 2,3,5- triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue. RESULTS: When compared with the control group, 10-1 treated mice showed significantly lower mNDS scores (p = 0.000336) and incidence of circling (p = 0.00256). Calculated infarct volumes, based on TTC staining, of the 10-1 treated group were significantly lower (p = 0.0009) when compared to the control group. CONCLUSIONS: We have shown that 10-1 is a promising therapeutic agent and provides substantial neuroprotection through its many multicellular processes in a rodent study. Current studies are being conducted to investigate the mechanism of 10-1 as neuroprotectant.

453 Ketamine Promotes Sustained Brain-Derived Neurotrophic Factor Levels in the Corticoaccumbens Circuit

OBJECTIVES/GOALS: Neuropsychiatric disorders classified as synaptopathies are marked by a glutamate-associated hypofrontality which impacts decision making and impulsivity. We hypothesized that behavioral efficacy of the psychoplastogen ketamine is mediated in part through lasting promotion of markers of synaptic strength in corticoaccumbens circuit. METHODS/STUDY POPULATION: Male, Sprague-Dawley rats received an intraperitoneal (i.p.) injection of saline, single ketamine (10 mg/kg; 1x/day), or repeated ketamine (10 mg/kg; 1x/day for three days). Twenty-four hrs following the dosing regimen, animals were euthanized, and brains dissected to harvest corticoaccumbens structures including the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). mRNA was extracted and converted to cDNA. Levels of brain derived neurotrophic factor (BDNF) exon II mRNA were quantified using reverse transcriptase polymerase chain reaction (RT-PCR); cyclophilin A (PPIA) was used as a loading control. Gene expression differences in ketamine-treated rats were identified versus saline-treated rats. BDNF protein levels were quantified using capillary-electrophoresis immunoblotting. RESULTS/ANTICIPATED RESULTS: Repeated, but not single, ketamine administration decreased mPFC, but increased NAc, BDNF exon II mRNA levels versus saline (p&lt;0.05). Single and repeated ketamine administration increased NAC BDNF protein (p&lt;0.05), while both dosing paradigms induced a trend towards an increase in mPFC BDNF levels. DISCUSSION/SIGNIFICANCE: We discovered a dosing regimen-dependent and sustained effects of ketamine administration on BDNF levels in the rodent brain. Taken together, ketamine-mediated BDNF levels may sustain synaptic strengthening mechanisms supporting future investigation into the utility of ketamine for diseases characterized by synaptopathies.

Halofuginone prevents outer retinal degeneration in a mouse model of light-induced retinopathy.

Photoreceptor cell death can cause progressive and irreversible visual impairments. Still, effective therapies on retinal neuroprotection are not available. Hypoxia-inducible factors (HIFs) are transcriptional factors which strongly regulate angiogenesis, erythropoiesis, intracellular metabolism, and programed cell death under a hypoxic or an abnormal metabolic oxidative stress condition. Therefore, we aimed to unravel that inhibition of HIFs could prevent disease progression in photoreceptor cell death, as recent studies showed that HIFs might be pathologic factors in retinal diseases. Adult male balb/cAJcl (8 weeks old; BALB/c) were used to investigate preventive effects of a novel HIF inhibitor halofuginone (HF) on a murine model of light-induced retinopathy. After intraperitoneal injections of phosphate-buffered saline (PBS) or HF (0.4 mg/kg in PBS) for 5 days, male BALB/c mice were subjected to a dark-adaption to being exposed to a white LED light source at an intensity of 3,000 lux for 1 hour in order to induce light-induced retinal damage. After extensive light exposure, retinal damage was evaluated using electroretinography (ERG), optical coherence tomography (OCT), and TUNEL assay. Light-induced retinal dysfunction was suppressed by HF administration. The amplitudes of scotopic a-wave and b-wave as well as that of photopic b-wave were preserved in the HF-administered retina. Outer retinal thinning after extensive light exposure was suppressed by HF administration. Based on the TUNEL assay, cell death in the outer retina was seen after light exposure. However, its cell death was not detected in the HF-administered retina. Halofuginone was found to exert preventive effects on light-induced outer retinal cell death.

Effect of rifampicin on TLR4-signaling pathways in the nucleus accumbens of the rat brain during abstinence of long-term alcohol treatment.

The treatment with the antibiotic rifampicin (Rif) led to a decrease in the frequency of neurodegenerative pathologies. There are suggestions that the mechanism of action of Rif may be mediated by its effect on toll-like receptor (TLR)4-dependent pathways. We evaluated the expression status of TLR4-dependent genes during abstinence from long-term alcohol treatments in the nucleus accumbens (NAc) of the rat brain, and also studied the effects of Rif to correct these changes. The long-term alcohol treatment was performed by intragastric delivery of ethanol solution. At the end of alcohol treatment intraperitoneal injections of Rif (100mg/kg) or saline were made. Extraction of the brain structures was performed on the 10th day of abstinence from alcohol. We used the SYBR Green qPCR method to quantitatively analyze the relative expression levels of the studied genes. The long-term alcohol treatment promotes an increase in the level of TLR4 mRNA and mRNA of its endogenous ligand high-mobility group protein B1 during abstinence drop alcohol in NAc of rats. The use of Rif in our study led to a decrease in the increased expression of high-mobility group protein B1, Tlr4, and proinflammatory cytokine genes (Il1β, Il6) in the NAc of the rat brain during abstinence of long-term alcohol treatment. In addition, Rif administration increased the decreased mRNA levels of anti-inflammatory cytokines (Il10, Il11). The data obtained indicate the ability of Rif to correct the mechanisms of the TLR4 system genes in the NAc of the rat brain during alcohol abstinence.

Ovarian lavage / artificial insemination method as an alternative approach for controlled reproduction of the northern pike (Esox lucius)

This study aimed to compare the effectiveness of the ovarian lavage / artificial insemination method with the traditional hormonal administration and fertilization methods over the artificial reproduction of the northern pike (Esox lucius). For this purpose, groups of five females were treated as follows: intraperitoneal injection of saline (C1); ovarian lavage with saline (C2); intraperitoneal injection of carp pituitary extract (CPE, T1); ovarian lavage with CPE (T2); intraperitoneal injection of CPE and ovarian lavage with semen after 72 h (T3); ovarian lavage with CPE and ovarian lavage with semen after 72 h (T4). According to the results, no fish ovulated in the control groups (C1 and C2). There were no significant differences (n.s.) among experimental treatments (P > 0.05, n.s.) in the reproductive parameters, such as latency time, ovulation rate, stripped egg amount, and pseudo-gonadosomatic index. The lowest fertilization rate (54.8%) was observed in the T4 treatment and significantly differed from the T1 and T2 treatments (P < 0.05). Moreover, the highest survival at swim-up stage was measured in the T4 treatment with a significant difference compared to the T1 group (P < 0.05). The survival at the eyed-egg stage, hatching rate, and malformations were similar (P > 0.05, n.s.) in all applied strategies. The results demonstrated that the ovarian lavage / artificial insemination method could be applied to control northern pike reproduction, like the traditional fertilization method. Consequently, this novel technique can be suggested as an alternative strategy to facilitate the hatchery operations in the controlled reproduction of this species.

Downregulation of lncRNA CDKN2B-AS1 attenuates inflammatory response in mice with allergic rhinitis by regulating miR-98-5p/SOCS1 axis

Long non-coding RNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in various diseases has been verified. However, the underlying mechanism of CDKN2B-AS1 contributes to the development of allergic rhinitis (AR) remains unknown. To evaluate the impact of CDKN2B-AS1 on AR, BALB/c mice were sensitized by intraperitoneal injection of normal saline containing ovalbumin (OVA) and calmogastrin to establish an AR model. Nasal rubbing and sneezing were documented after the final OVA treatment. The concentrations of IgE, IgG1, and inflammatory elements were quantified using ELISA. Hematoxylin and eosin (H&E) staining and immunofluorescence were used to assess histopathological variations and tryptase expression, respectively. StarBase, TargetScan and luciferase reporter assays were applied to predict and confirm the interactions among CDKN2B-AS1, miR-98-5p, and SOCS1. CDKN2B-AS1, miR-98-5p, and SOCS1 levels were assessed by quantitative real-time PCR (qRT-PCR) or western blotting. Our results revealed that CDKN2B-AS1 was obviously over-expressed in the nasal mucosa of AR patients and AR mice. Down-regulation of CDKN2B-AS1 significantly decreased nasal rubbing and sneezing frequencies, IgE and IgG1 concentrations, and cytokine levels. Furthermore, down-regulation of CDKN2B-AS1 also relieved the pathological changes in the nasal mucosa, and the infiltration of eosinophils and mast cells. Importantly, these results were reversed by the miR-98-5p inhibitor, whereas miR-98-5p directly targeted CDKN2B-AS1, and miR-98-5p negatively regulated SOCS1 level. Our findings demonstrate that down-regulation of CDKN2B-AS1 improves allergic inflammation and symptoms in a murine model of AR through the miR-98-5p/SOCS1 axis, which provides new insights into the latent functions of CDKN2B-AS1 in AR treatment.

Attenuated incubation of ethanol-induced conditioned taste aversion in a model of dependence.

Preclinical studies report attenuated ethanol-induced conditioned taste aversion (CTA) following chronic ethanol exposure, suggesting that tolerance develops to the aversive properties of ethanol. However, these studies are confounded by pre-exposure to the unconditioned stimulus (US; ethanol), which is well known to hinder conditioning. This study was designed to determine whether chronic ethanol exposure produces tolerance to the aversive properties of ethanol in the absence of a US pre-exposure confound. CTA was performed in adult male and female Long-Evans rats by pairing 0.1% ingested saccharin with an intraperitoneal injection of ethanol (1.5 or 2.0g/kg) or saline. Rats were then rendered ethanol dependent using chronic intermittent ethanol (CIE) vapor exposure. Controls were exposed to room air (AIR). The effect of chronic ethanol on CTA expression and reconditioning were examined following vapor exposure. Prior to vapor exposure, both sexes developed CTA to a comparable degree with 2.0g/kg producing greater CTA than 1.5g/kg ethanol. Following vapor exposure, AIR controls exhibited an increase in CTA magnitude compared to pre-vapor levels. This effect was largely absent in CIE-exposed rats. Re-conditioning after vapor exposure facilitated increased CTA magnitude to a similar degree in AIR- and CIE-exposed males. In contrast, CTA magnitude was unchanged by re-conditioning in females. These data suggest that chronic ethanol does not facilitate tolerance to the aversive properties of ethanol but rather attenuates incubation of ethanol-induced CTA. Loss of CTA incubation suggests that CIE exposure disrupts circuits encoding aversion.

Doxycycline reversal of amphetamine-induced mania-like behavior is related to adjusting brain monoamine abnormalities and antioxidant effects in primary hippocampal neurons.

Mania is associated with disturbed dopaminergic transmission in frontotemporal regions. D-amphetamine (AMPH) causes increased extracellular DA levels, considered an acknowledged mania model in rodents. Doxycycline (DOXY) is a second-generation tetracycline with promising neuroprotective properties. Here, we tested the hypothesis that DOXY alone or combined with Lithium (Li) could reverse AMPH-induced mania-like behavioral alterations in mice by the modulation of monoamine levels in brain areas related to mood regulation, as well as cytoprotective and antioxidant effects in hippocampal neurons. Male Swiss mice received AMPH or saline intraperitoneal (IP) injections for 14 days. Between days 8-14, mice receive further IP doses of DOXY, Li, or their combination. For in vitro studies, we exposed hippocampal neurons to DOXY in the presence or absence of AMPH. DOXY alone or combined with Li reversed AMPH-induced risk-taking behavior and hyperlocomotion. DOXY also reversed AMPH-induced hippocampal and striatal hyperdopaminergia. In AMPH-exposed hippocampal neurons, DOXY alone and combined with Li presented cytoprotective and antioxidant effects, while DOXY+Li also increased the expression of phospho-Ser133-CREB. Our results add novel evidence for DOXY's ability to reverse mania-like features while revealing that antidopaminergic activity in some brain areas, such as the hippocampus and striatum, as well as hippocampal cytoprotective effects may account for this drug's antimanic action. This study provides additional rationale for designing clinical trials investigating its potential as a mood stabilizer agent.

Protective Effects of Xanthine Derivatives Against Arsenic Trioxide-Induced Oxidative Stress in Mouse Hepatic and Renal Tissues.

In this study, the protective efficacy of pentoxifylline (PTX) as a xanthine derivative against arsenic trioxide (ATO)-induced kidney and liver damage in mice was investigated. Thirty-six mice were divided into six groups, receiving intraperitoneal injections of saline, ATO, PTX, or a combination for four weeks. Blood samples were analyzed for serum biochemistry, while hepatic tissue underwent examination for histopathological changes and assessment of oxidative stress markers and antioxidant gene expression through Real-Time PCR. ATO exposure significantly increased serum markers (creatinine, ALT, BUN, ALP, AST) and induced histopathological changes in the liver. Moreover, it elevated renal and hepatic nitric oxide (NO) and lipid peroxidation (LPO) levels, and reduced antioxidant enzyme expression (CAT, GSR, GPx, MPO, SOD), total thiol groups (TTGs), and total antioxidant capacity (TAC). Conversely, PTX treatment effectively lowered serum hepatic and renal markers, improved antioxidant markers, and induced histopathological alterations. Notably, PTX did not significantly affect renal and hepatic NO levels. These findings suggest that PTX offers therapeutic potential in mitigating liver and acute kidney injuries induced by various insults, including exposure to ATO.