Saline-treated Mice Research Articles

Convection-enhanced delivery of [177Lu]Lu-labeled gold nanoparticles combined with anti-PD1 checkpoint immunotherapy improves the survival of immunocompetent C57BL/6J mice with orthotopic GL261 murine glioma tumors

IntroductionOur objective was to study convection enhanced delivery (CED) of 177Lu-labeled metal chelating polymer (MCP) conjugated to gold nanoparticles ([177Lu]Lu-MCP-AuNP) alone or combined with anti-PD1 immune checkpoint inhibition (ICI) for improving the survival of immunocompetent C57BL/6J mice with orthotopic GL261 murine glioma tumors. MethodsC57BL/6J mice with GL261 tumors were treated with [177Lu]Lu-MCP-AuNP (0.8 or 2.7 MBq; 4 × 1011 AuNP) alone or combined with anti-PD1 antibodies (200 μg i.p. every 2 d × 3 doses). Control mice received normal saline, non-radioactive MCP-AuNP or anti-PD1 antibodies. Kaplan-Meier median survival was estimated. T-cell infiltration into the brain was probed by flow cytometry. Toxicity was assessed by monitoring body weight and cognitive function tests [Object Location Test (OLT) and Novel Object Recognition Test (NORT)] and T2-weighted MRI of the brain, overall health and ex vivo histopathological examination of the brain. ResultsTreatment with [177Lu]Lu-MCP-AuNP (0.8 MBq) significantly increased median survival compared to MCP-AuNP (29 vs. 25 d; P = 0.007) or normal saline-treated mice (24 d; P < 0.001). Combining [177Lu]Lu-MCP-AuNP (0.8 MBq) with anti-PD1 antibodies increased median survival to 32 d (P < 0.0001 vs. normal saline). Increasing the mean amount of [177Lu]Lu-MCP-AuNP to 2.7 MBq and combining with anti-PD1 antibodies extended survival to at least 218 d in 5/9 mice. Increased CD8+ cytotoxic T-cells and decreased CD4+ helper T-cells were found in the brain vs. normal saline-treated mice. No weight loss (>20 %) was observed for treated or control mice. There was no change in cognitive function in mice treated with [177Lu]Lu-MCP-AuNP (0.8 MBq) alone or combined with anti-PD1 antibodies assessed by the OLT or NORT. T2-weighted MRI in mice treated with 2.7 MBq [177Lu]Lu-MCP-AuNP combined with anti-PD1 antibodies revealed edema, gliosis and ex vacuo dilatation of the ventricle proximal to the site of infusion. Histopathological examination of the brain revealed dilatation of the ventricle and gliosis proximal to the site of infusion but no radiation necrosis. MRI and histological analysis did not reveal tumor in the brain of these mice. Mice treated with 2.7 MBq [177Lu]Lu-MCP-AuNP combined with anti-PD1 antibodies did not demonstrate overall deleterious health effects. ConclusionsWe conclude that CED of [177Lu]Lu-MCP-AuNP combined with anti-PD1 checkpoint immunotherapy improved the survival of immunocompetent C67BL/6J mice with GL261 glioma tumors in the brain. Higher administered amounts of [177Lu]Lu-MCP-AuNP (2.7 MBq vs. 0.8 MBq) were most effective and yielded long-term survival. Advances in knowledge and implications for patient careThis study demonstrates that combining a locally-infused radiation nanomedicine, [177Lu]Lu-MCP-AuNP and anti-PD1 checkpoint immunotherapy improved the survival of mice with glioma tumors in the brain. In the future, this treatment may be useful to treat residual tumor at the surgical margins in patients with GBM to prevent local recurrence and improve survival.

Social Avoidance of Mice in Pain in Naturalistic Conditions

AbstractPain and social behavior are subject to reciprocal modulation. Social animals show attenuated pain behavior in the presence of conspecifics, while observers are in turn affected by exposure to another individual in pain. Both phenomena have been established in rodents, which in addition to experiencing emotional contagion from afflicted conspecifics may act to relieve their afflicted state. Little has been done to investigate the motivation of such prosocial behavior in naturalistic conditions. Here, using a novel formalin test targeting the nape of the neck, a socially relevant area, we investigated nocifensive behaviors and social interactions in mice group-housed in a seminatural environment (SNE). In the SNE, formalin-injected mice displayed fewer back-scratching occurrences than when housed alone, which was inversely correlated to the social behavior received. These mice also emitted and received fewer social interactions, attesting of social withdrawal. With respect to dyadic exchanges, saline-treated mice initiated fewer allosniffing and anogenital sniffing episodes towards formalin-treated mice than towards other saline-treated ones. These findings are counter to those showing empathy and prosocial approach in mouse pain models. It is possible that in naturalistic conditions that allow the mice to express a wide range of their behavioral repertoire, healthy mice simply avoid individuals in pain and the cost associated with emotional contagion. Interestingly, behaviors involving direct body contact, namely allogrooming and pair-resting, were not different between saline- and formalin-treated mice and thus may carry a prosocial, altruistic component. These findings unveil new patterns of social modulation by pain in a naturalistic laboratory setting holding high translational value.

Innate Immune Reprogramming of Macrophages in the Parous Murine Mammary Gland

Uncomplicated childbirth is associated with an increased risk of developing breast cancer (BC) five-to-ten years postpartum (postpartum breast cancer; PPBC) compared with age-matched nulliparous individuals. PPBC is also associated with a poor prognosis relative to nulliparous individuals with BC. Contrastingly, preeclampsia, an inflammatory pregnancy complication, protects against PPBC development. We hypothesize that differential parity-induced innate immune reprogramming of mammary gland macrophages may be associated with parity-associated increased PPBC risk and preeclampsia-associated protection against PPBC. Female BALB/c mice were mated with male C57Bl/6 mice to establish allogeneic pregnancies. On gestational day (GD) 10.5, dams were injected with saline (controls) or lipopolysaccharide (LPS; 20 μg/kg) to induce inflammatory pregnancy complications. Age-matched nulliparous controls also received LPS or saline. Following birth, parous mice were force-weaned on postnatal day (PD) 7 to induce synchronous involution. Macrophages were isolated from mammary glands upon euthanasia in pregnancy (GD 14.5), lactation (PD 7), involution (PD 10) and regression (PD 28), plated (4x104 cells/well) and exposed to a 23-hour secondary challenge (LPS; 100 ng/ml; Pam3Cys; 10 µg/ml). Cytokine concentrations in cell culture supernatant were assessed using a multiplex assay (Eve Technologies) and data were analyzed by two-way ANOVA with Tukey’s posthoc test. During involution, compared with supernatant from mammary gland macrophages of age-matched nulliparous saline-treated mice, supernatant from macrophages from parous saline-treated mice exhibited significantly increased interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), and significantly decreased IL-6 following secondary challenge. TNF-α, IL-6 and IL-10 in supernatant from macrophages from parous LPS-treated mice was significantly decreased compared with parous saline-treated controls. Analysis of data collected during pregnancy, lactation and regression is ongoing to temporally evaluate reprogramming. These preliminary results demonstrate differential reprogramming in mammary gland macrophages relative to parity and inflammatory pregnancy complication status and rationalize understanding contributions of completion of a prior pregnancy on PPBC risk and progression.

Abstract Mo007: Molecular Basis of Sepsis-Induced Cardiomyopathy Under Anthracycline Treatment: Insights from Mouse Model

Anthracyclines constitute a fundamental component in numerous paediatric chemotherapy regimens. However, its myelosuppressive &amp; cardiotoxic nature put patients at risk of sepsis and sepsis-induced cardiomyopathy (SIC). When anthracycline-treated patients develop SIC, successful treatment of sepsis is sufficient for the restoration of cardiac function, giving rise to the 'transient cardiac dysfunction' phenomenon. Although transient it poses compromised clinical outcomes &amp; the mechanism driving this phenomenon remains elusive. We hypothesise that the molecular regulators of the phenomenon lie within cardiac cell populations &amp; is controlled by transcriptional activation. Thus, we aim to model 'transient cardiac dysfunction' in mice and conduct transcriptomics on their heart tissues. ‘Transient cardiac dysfunction’ was modelled in male juvenile C57BL6 mice treated with a cumulative of 10mg/kg doxorubicin followed by sepsis induction via cecal ligation &amp; perforation (CLP) to induce SIC. To recapitulate ‘transient cardiac dysfunction’, antibiotics were administered 20h post-CLP to treat sepsis &amp; restore cardiac function. Controls against doxorubicin (saline treatment) was established. Echocardiography and biological sample curation were conducted at pre-SIC (before CLP), onset-SIC (10h post-CLP) &amp; post-SIC (60h post-CLP). Sepsis was verified based on procalcitonin fold change as compared to pre-SIC (n=3-6). Procalcitonin was 15±1.6 folds higher in dox- (n=3) &amp; 10±0.39 folds higher in saline-treated (n=3) mice at onset-SIC. The infection was more aggressive in dox-treated mice as procalcitonin remained elevated at post-SIC (17.1±2.0, n=4), whereas in saline-treated mice, procalcitonin was reduced by post-SIC (7.9±1.0, n=3). In saline-treated mice, LVEF(%) was reduced at onset-SIC (49.0%±3.0, n=7) from pre-SIC (58.1±1.1, n=7) and recovered to baseline by post-SIC (61.8±1.7, n=5). In the contrary, LVEF in dox-treated mice (n=7) was increased at onset-SIC (62.5±1.7) from pre-SIC (56.0±0.8) &amp; sustained till post-SIC (60.1±1.0), suggesting cardioprotection. Normal distribution was verified before statistical hypothesis testing. In conclusion, ‘transient cardiac dysfunction’ was modelled in saline-treated mice, &amp; novel cardio protective effect was observed in dox-treated mice. Moving forward, longitudinal snRNA-seq will be conducted on whole hearts to identify cardiac cell populations &amp; genes responsible for ‘transient cardiac dysfunction’ &amp; cardioprotection.

CpG oligonucleotides induce acute murine thrombocytopenia dependent on toll-like receptor 9 and spleen tyrosine kinase pathways

BackgroundCpG oligonucleotides (ODNs) are synthetic single-stranded DNA sequences that act as immunostimulants. They have been increasingly used to treat several cancers; however, thrombocytopenia is a potential recognized side effect of some sequences. ObjectivesWe tested the ability of 2 CpG ODNs (ODN 2395 and ISIS 120704) to induce thrombocytopenia when administered to BALB/c mice and determined mechanisms associated with thrombocytopenia. MethodsBALB/c mice were prebled and then injected with titrated doses of CpG ODNs, and platelet counts were determined. The mice were treated with intravenous immunoglobulin (IVIg) or various inhibitors and antagonists of toll-like receptor 9 (TLR9) and spleen tyrosine kinase (Syk) to determine their effects on thrombocytopenia. ResultsCompared with saline-treated mice or mice treated with 2′-O-methoxyethyl–modified antisense ODN, both ODN 2395 and ISIS 120704 induced acute dose-dependent thrombocytopenia within 3 and 24 hours, respectively. The thrombocytopenia was associated with significant increases in plasma monocyte chemoattractant protein 1. IVIg administration significantly rescued the CpG ODN–induced thrombocytopenia, as did treatment with either a Syk inhibitor or TLR9 antagonists. In vitro, CpG ODN could activate human platelets and this correlated significantly with enhanced IVIg- and Syk-dependent phagocytosis by THP-1 monocytes. ConclusionThese results suggest that CpG ODNs induce acute inflammatory-associated (IVIg-sensitive) thrombocytopenia that can be alleviated by Syk- or TLR9-blockade, and an IVIg- and Syk-dependent platelet clearance pathway appears primarily responsible for the thrombocytopenia.

The presence of circulating human apolipoprotein J reduces the occurrence of cerebral microbleeds in a transgenic mouse model with cerebral amyloid angiopathy

BackgroundCerebral amyloid angiopathy (CAA) is characterized by amyloid-β (Aβ) deposition in cerebral vessels, leading to lobar cerebral microbleeds (CMB) and intracerebral hemorrhages (ICH). Apolipoprotein J (ApoJ) is a multifunctional chaperone related to Aβ aggregation and clearance. Our study investigated the vascular impact of chronic recombinant human Apolipoprotein J (rhApoJ) treatment in a transgenic mouse model of β-amyloidosis with prominent CAA.MethodsTwenty-month-old APP23 C57BL/6 mice received 25 doses of rhApoJ (1 mg/kg) (n = 9) or saline (n = 8) intraperitoneally for 13 weeks, while Wild-type (WT) mice received saline (n = 13). Postmortem brains underwent T2*-weighted magnetic resonance imaging (MRI) to detect hemorrhagic lesions. Aβ levels and distribution, cerebral fibrinogen leakage, brain smooth muscle actin (sma), and plasma matrix metalloproteinases and inflammatory markers were analyzed after treatments. Additionally, plasma samples from 22 patients with lobar ICH were examined to determine the clinical relevance of the preclinical findings.ResultsrhApoJ-treated APP23 presented fewer cortical CMBs (50–300 μm diameter) (p = 0.012) and cortical larger hemorrhages (> 300 μm) (p = 0.002) than saline-treated mice, independently of Aβ brain levels. MRI-detected hemorrhagic lesions correlated with fibrinogen cerebral extravasation (p = 0.011). Additionally, rhApoJ-treated mice presented higher number of sma-positive vessels than saline-treated mice (p = 0.038). In rhApoJ-treated mice, human ApoJ was detected in plasma and in occasional leptomeningeal vessels, but not in the parenchyma, suggesting that its mechanism of action operates through the periphery. The administration of rhApoJ induced an increase in plasma Groα (p = 0.035) and MIP-1α (p = 0.035) levels, while lower MMP-12 (p = 0.046) levels, compared to the saline-treated group. In acute lobar ICH patients, MMP-12 plasma levels correlated with larger hemorrhage volume (p = 0.040) and irregular ICH shape (p = 0.036).ConclusionsChronic rhApoJ treatment in aged APP23 mice ameliorated CAA-related neurovascular damage by reducing the occurrence of CMB. We propose that rhApoJ may prevent blood-brain barrier (BBB) leakage and CMB appearance partly through circulating MMP-12 modulation.

Viscerosensory signalling to the nucleus accumbens via the solitary tract nucleus.

The nucleus of the solitary tract (NTS) receives direct viscerosensory vagal afferent input that drives autonomic reflexes, neuroendocrine function and modulates behaviour. A subpopulation of NTS neurons project to the nucleus accumbens (NAc); however, the function of this NTS-NAc pathway remains unknown. A combination of neuroanatomical tracing, slice electrophysiology and fibre photometry was used in mice and/or rats to determine how NTS-NAc neurons fit within the viscerosensory network. NTS-NAc projection neurons are predominantly located in the medial and caudal portions of the NTS with 54 ± 7% (mice) and 65 ± 3% (rat) being TH-positive, representing the A2 NTS cell group. In horizontal brainstem slices, solitary tract (ST) stimulation evoked excitatory post-synaptic currents (EPSCs) in NTS-NAc projection neurons. The majority (75%) received low-jitter, zero-failure EPSCs characteristic of monosynaptic ST afferent input that identifies them as second order to primary sensory neurons. We then examined whether NTS-NAc neurons respond to cholecystokinin (CCK, 20 μg/kg ip) invivo in both mice and rats. Surprisingly, there was no difference in the number of activated NTS-NAc cells between CCK and saline-treated mice. In rats, just 6% of NTS-NAc cells were recruited by CCK. As NTS TH neurons are the primary source for NAc noradrenaline, we measured noradrenaline release in the NAc and showed that NAc noradrenaline levels declined in response to cue-induced reward retrieval but not foot shock. Combined, these findings suggest that high-fidelity afferent information from viscerosensory afferents reaches the NAc. These signals are likely unrelated to CCK-sensitive vagal afferents but could interact with other sensory and higher order inputs to modulate learned appetitive behaviours.

Hypersensitivity of the nicotinic acetylcholine receptor subunit (CHRNA2L9′S/L9′S) in female adolescent mice produces deficits in nicotine-induced facilitation of hippocampal-dependent learning and memory

Adolescence is characterized by a critical period of maturation and growth, during which regions of the brain are vulnerable to long-lasting cognitive disturbances. Adolescent exposure to nicotine can lead to deleterious neurological and psychological outcomes. Moreover, the nicotinic acetylcholine receptor (nAChR) has been shown to play a functionally distinct role in the development of the adolescent brain. CHRNA2 encodes for the α2 subunit of nicotinic acetylcholine receptors associated with CA1 oriens lacunosum moleculare GABAergic interneurons and is associated with learning and memory. Previously, we found that adolescent male hypersensitive CHRNA2L9′S/L9′ mice had impairments in learning and memory during a pre-exposure-dependent contextual fear conditioning task that could be rescued by low-dose nicotine exposure. In this study, we assessed learning and memory in female adolescent hypersensitive CHRNA2L9′S/L9′ mice exposed to saline or a subthreshold dose of nicotine using a hippocampus-dependent task of pre-exposure-dependent contextual fear conditioning. We found that nicotine-treated wild-type female mice had significantly greater improvements in learning and memory than both saline-treated wild-type mice and nicotine-treated CHRNA2L9′S/L9′ female mice. Thus, hyperexcitability of CHRNA2 in female adolescent mice ablated the nicotine-mediated potentiation of learning and memory seen in wild-types. Our results indicate that nicotine exposure during adolescence mediates sexually dimorphic patterns of learning and memory, with wild-type female adolescents being more susceptible to the effects of sub-threshold nicotine exposure. To understand the mechanism underlying sexually dimorphic behavior between hyperexcitable CHRNA2 mice, it is critical that further research be conducted.

Inhibition of somatostatin enhances the long-term metabolic outcomes of sleeve gastrectomy in mice

ObjectiveBariatric surgery is an effective treatment to obesity, leading to weight loss and improvement in glycemia, that is characterized by hypersecretion of gastrointestinal hormones. However, weight regain and relapse of hyperglycemia are not uncommon. We set to identify mechanisms that can enhance gastrointestinal hormonal secretion following surgery to sustain weight loss. MethodsWe investigated the effect of somatostatin (Sst) inhibition on the outcomes of bariatric surgery using a mouse model of sleeve gastrectomy (SG). ResultsSst knockout (sst-ko) mice fed with a calorie-rich diet gained weight normally and had a mild favorable metabolic phenotype compared to heterozygous sibling controls, including elevated plasma levels of GLP-1. Mathematical modeling of the feedback inhibition between Sst and GLP-1 showed that Sst exerts its maximal effect on GLP-1 under conditions of high hormonal stimulation, such as following SG. Obese sst-ko mice that underwent SG had higher levels of GLP-1 compared with heterozygous SG-operated controls. The SG-sst-ko mice regained less weight than controls and maintained lower glycemia months after surgery. Obese wild-type mice that underwent SG and were treated daily with a Sst receptor inhibitor for two months had higher GLP-1 levels, regained less weight, and improved metabolic profile compared to saline-treated SG-operated controls, and compared to inhibitor or saline-treated sham-operated obese mice. ConclusionsOur results suggest that inhibition of Sst signaling enhances the long-term favorable metabolic outcomes of bariatric surgery.

Trauma-Induced Vestibular Dysfunction: Improved Repair Under Local Treatment With α1-Antitrypsin.

To characterize vestibular recovery in a mouse model of unilateral labyrinthotomy under local AAT and dexamethasone treatment. Alpha1-antitrypsin (AAT) is a circulating tissue-protective molecule that rises during inflammatory conditions and promotes inflammatory resolution. Its local concentration in human perilymph inversely correlates with the severity of inner ear dysfunction; concomitantly, mice that overexpress AAT and undergo inner ear trauma rapidly restore vestibular function. Locally applied AAT has yet to be examined in this context, nor has it been directly compared with anti-inflammatory corticosteroid treatment. Wild-type mice C57BL/6 underwent a unilateral inner ear injury. Nine microliters of saline, clinical-grade AAT (180 μg/site), dexamethasone (4 mg/site), or both were applied locally on Days 0, 1, and 2 (n = 5/group). Vestibular function was assessed for 7 days. An in vitro human epithelial gap closure assay was performed using A549 cells in the presence of AAT and/or dexamethasone. Upon labyrinthotomy, all groups displayed severe vestibular dysfunction. Saline-treated mice showed the longest impairment. That group and the dexamethasone group displayed partial to no recovery, while AAT-treated mice exhibited complete recovery within 7 days; at this time point, dexamethasone-treated mice exhibited 50% recovery. Objective vestibular testing showed similar outcomes. In vitro, cotreatment with AAT and dexamethasone resulted in a gap closure dynamic that was superior to AAT alone at 6 h and superior to DEX alone at 48 h. Locally applied AAT treatment is superior to locally applied dexamethasone in promoting vestibular recovery in vivo. Ongoing studies are exploring the potential advantages of AAT combined with early low-dose dexamethasone therapy.

Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry–based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

Left Ventricular Dilation Resulting from the Absence of Either Rgs2 or Rgs5 Is Associated with Increased Immune Cell Infiltration in the Myocardium

Sustained sympathoexcitation is a hallmark of heart failure, with equally sustained activation of downstream signaling pathways involving Gs- and Gi/o-coupled β-adrenergic receptors (βAR) stimulating cAMP production and PKA activation. A critical mechanism controlling G protein-mediated βAR signaling is the activity of regulator of G protein signaling (RGS) proteins, including members of the B/R4 subfamily, which accelerate the termination of Gq/11 and Gi/o protein signaling. Previous studies by us and others reported that moderation of Gi/o activity separately by RGS2 and RGS5 of the R4 subfamily is key to maintaining normal ventricular rhythm. The loss of one or both R4 RGS proteins also results in left ventricular (LV) dilation and reduced ejection fraction. Here, we tested the hypothesis that the LV structural remodeling is due to the disruption of the coordinated activity of RGS2 and 5 resulting in sensitization of βAR signaling. To test this hypothesis, we challenged wild type (WT), Rgs2 KO ,Rgs5 KO, and Rgs2/5 double knockout (dbKO) adult male mice with sub-chronic infusion of isoproterenol (ISO, 30 mg/kg/day, 3 days) via a peritoneal osmotic minipump implantation. Rgs2 KO and Rgs5 KO mice showed increased LV chamber size at baseline, which was unchanged by ISO administration. In saline-treated animals, the loss of Rgs2 increased Rgs5 mRNA expression, while Rgs5 absence increased Rgs4 mRNA expression. ISO infusion induced concentric cardiac hypertrophy in WT mice, as evidenced by increased heart weight/tibia length (HW/TL) ratio and cardiomyocyte cross sectional area. While ISO infusion increased interstitial fibrosis in midventricular tissue and HW/TL ratio in dbKO mice, it did not affect cardiomyocyte cross sectional area or exacerbate LV dilation, despite increased expression of the cardiac fetal gene Nppa as well as the sarcomeric protein α-actinin and Ca2+/calmodulin-dependent kinase II (CaMKII). Cardiact tissue from saline-treated Rgs2 KO and Rgs5 KO mice had markedly increased CD45+ cells vs WT, while tissue from Rgs5 KO mice showed increased CD68+ cells, as revealed by immunohistochemistry. Taken together, these results indicate that ventricular remodeling seen in Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO mice is insensitive to sustained βAR stimulation but may be due to increased cardiac resident immune cell population. This work was supported by NIH R56 DK132859-01A1 and R01 HL139754, and Craig H. Neilsen Foundation Grant 382566 to Patrick Osei-Owusu. 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.

Low Intensity Renal Ultrasound Exposure Reduces Systemic Inflammation Induced by LPS Challenge in Mice

Low-intensity pulsed ultrasound (LIPUS), a procedure historically used to stimulate bone healing and tissue repair, activates the splenic cholinergic anti-inflammatory pathway and ultimately reduces circulating and tissue-specific pro-inflammatory cytokine release if applied to the kidney prior to renal ischemia/reperfusion injury. We questioned whether LIPUS would be effective in reducing the inflammatory response to lipopolysaccharide (LPS) if exposure occurred as pre- or post-treatment. We hypothesized that LIPUS exposure before an LPS challenge would suppress the subsequent inflammatory response in mice, and that LIPUS exposure after an LPS challenge would be similarly beneficial. Female C57BL/6J mice were randomized into Saline, LPS, LPS/Pre-treatment and LPS/Post-treatment groups at 15 weeks of age. Pre-treated mice were subjected to 20-minute LIPUS exposure while under anesthesia for five consecutive days. On the 6th day, mice were injected with LPS (1 mg/kg dissolved in saline, IP) or saline (100 μL). One hour after LPS injection, post-treated mice were exposed to LIPUS for 20 min. Three hours after LPS or saline injection, all mice were humanely euthanized, and plasma, spleen and kidney were harvested for analysis of proinflammatory cytokines associated with the response to LPS. LPS significantly increased plasma TNF-α (280±37 vs. 0.07±0.00 pg/mL), plasma IL-6 (96124±6688 vs. 425±181 pg/mL), splenic IL-6 (0.77±0.16 vs. 0.17±0.04 pg/μg of protein), and renal cortical IL-6 (0.50±0.07 vs. 0.01±0.00 pg/μg of protein) compared to saline-treated mice (all p&lt;0.05). LIPUS pre-treatment significantly reduced plasma TNF-α (183±15 pg/mL) and plasma IL-6 (63656±8441 pg/mL), without altering splenic and renal cytokines in LPS-treated mice. LIPUS post-treatment lowered plasma IL-6 (47432 ± 1182 pg/mL). In conclusion, five days of LIPUS exposure prior to an LPS challenge effectively reduced circulating inflammatory mediators, while a single LIPUS exposure after an LPS challenge also successfully reduced systemic IL-6. These findings demonstrate the potential in using LIPUS as a therapeutic strategy for quelling acute inflammatory responses and warrant further investigation of its use as a therapy for chronic inflammatory diseases. Studies funded by Department of Defense (LR210096) for KWM. 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.

PNA5, a Novel Glycosylated Angiotensin 1-7 Treatment Improves Infarct Size and Fibrotic Remodeling in Ischemic Reperfusion Injury

Background: Reperfusion during an acute ischemic event is of paramount importance to rescue cardiac tissue. Paradoxically, reperfusion of the ischemic tissue promotes further cardiac injury. Increased reactive oxidative species (ROS) and inflammation contribute to ischemic reperfusion injury (IR). PNA5, a glycosylated analog of angiotensin-(1-7), decreases ROS and inflammation. The purpose of this study is to indicate if PNA5 treatment has the ability to reduce infarct (IN) size and improve cardiac remodeling in IR. We hypothesize that PNA5 will improve fibrosis and diminish IN size in IR. Methods: 3-month-old male mice (C57BI/6J) underwent IR surgery. Mice were anesthetized and the left anterior descending artery was litigated for 45 minutes. Ischemic areas were then reperfused. Mice were immediately injected with PNA5 (subcutaneously s.c. at 100μg/kg, n=11) or with saline as control (n=11). Injections were continued daily with PNA5 or saline for 8-weeks. At the end of treatment, mice were sacrificed. Upon sacrifice, hearts were re-ligated and perfused with Evans Blue to identify the areas at risk (AAR). Hearts were stained with 2-3-4-triphenyltetrazolium chloride to show IN area. IN size and AAR were determined using Image J. Paraffn-embedded hearts were stained with Picrosirius Red for fibrosis. Fibrosis was measured as a ratio of collagen to total tissue area for 17 regions in the left ventricle (LV). Values are represented as mean ± standard error of mean. Results: PNA5-treated mice IN size was significantly decreased compared to saline-treated mice. (IN/AAR: IR-Saline 0.17± 0.02; PNA5 0.10± 0.01, p=0.005; IN/LV: IR-Saline 0.08± 0.01; PNA5 0.05±0.01; p=0.002, student’s T-test). PNA5 reduced fibrosis in the LV compared to IR-saline mice Conclusions: These results indicate PNA5 as a potential therapeutic treatment for IR as it improves IN size and fibrotic remodeling in IR. [Formula: see text] Supported by NIA U01AG066623 to MH. 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.

Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators.MethodsA male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators.ResultsSaline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1β) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks.ConclusionIntravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators.

C3aR in the medial prefrontal cortex modulates the susceptibility to LPS-induced depressive-like behaviors through glutamatergic neuronal excitability

Complement activation and prefrontal cortical dysfunction both contribute to the pathogenesis of major depressive disorder (MDD), but their interplay in MDD is unclear. We here studied the role of complement C3a receptor (C3aR) in the medial prefrontal cortex (mPFC) and its influence on depressive-like behaviors induced by systematic lipopolysaccharides (LPS) administration. C3aR knockout (KO) or intra-mPFC C3aR antagonism confers resilience, whereas C3aR expression in mPFC neurons makes KO mice susceptible to LPS-induced depressive-like behaviors. Importantly, the excitation and inhibition of mPFC neurons have opposing effects on depressive-like behaviors, aligning with increased and decreased excitability by C3aR deletion and activation in cortical neurons. In particular, inhibiting mPFC glutamatergic (mPFCGlu) neurons, the main neuronal subpopulation expresses C3aR, induces depressive-like behaviors in saline-treated WT and KO mice, but not in LPS-treated KO mice. Compared to hypoexcitable mPFCGlu neurons in LPS-treated WT mice, C3aR-null mPFCGlu neurons display hyperexcitability upon LPS treatment, and enhanced excitation of mPFCGlu neurons is anti-depressant, suggesting a protective role of C3aR deficiency in these circumstances. In conclusion, C3aR modulates susceptibility to LPS-induced depressive-like behaviors through mPFCGlu neuronal excitability. This study identifies C3aR as a pivotal intersection of complement activation, mPFC dysfunction, and depression and a promising therapeutic target for MDD.

Abstract 6030: Optimizing the therapeutic index of targeted α-particle radioimmuotherapy (TART) of HER2-positive breast cancer tumors in NRG mice with 225Ac-labeled trastuzumab

Abstract Introduction: Our aim was to optimize the therapeutic index of TART of HER2-positive BC in NRG mice by studying the effectiveness and normal tissue toxicity of trastuzumab IgG, F(ab’)2 or Fab modified with DOTA for complexing the α-particle emitter, 225Ac. Methods: The toxicity of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 and Fab were assessed in NRG mice (n=5) injected i.v. with 2 and 4 kBq (total 80 μg) separated by 8 d. Body weight was monitored and complete blood cell (CBC) counts and alanine aminotransferase (ALT) and creatinine (CRE) were measured at 14 d post-injection (p.i.). TART was performed in NRG mice (n=7) with s.c. HER2-positive 164/8-1B/H2N.luc+ xenografts injected i.v. with 2 and 4 kBq (total 80 μg) separated by 8 d of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 or Fab. Control mice received irrelevant [225Ac]Ac-DOTA-IgG, trastuzumab or saline. The tumor growth index (TGI=tumor volume/initial tumor volume) was measured and Kaplan-Meier median survival estimated. Tumor and normal tissue uptake (%ID/g) of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 and Fab (4 kBq) in tumor-bearing NRG mice were measured up to 14 d p.i.. Results: [225Ac]Ac-DOTA-trastuzumab F(ab’)2 and Fab caused no decrease in CBC, while [225Ac]Ac-DOTA-trastuzumab IgG decreased white blood cells by 4.5-fold, platelets by 7.5-fold, red blood cells by 1.2-fold and hematocrit by 1.2-fold compared to saline-treated mice. [225Ac]Ac-trastuzumab F(ab’)2 or Fab caused no increase in ALT or CRE. Body weight was not decreased in all groups of mice. [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 or Fab inhibited tumor growth (TGI at 15 d = 2.5, 1.8, and 1.9, respectively) vs. saline or trastuzumab (TGI= 6.3 and 5.2; P=0.0047 and 0.0028). Median survival was increased to 46 d for mice treated with [225Ac]Ac-DOTA-trastuzumab F(ab’)2 vs. 29 d for Fab (P=0.008), 22 d for IgG (P=0.0005) and 15 d for saline (P=0.0005). Median survival for mice treated with [225Ac]Ac-DOTA-IgG was 20 d and for trastuzumab was 22 d. Tumor uptake of [225Ac]Ac-DOTA-trastuzumab IgG and F(ab’)2 at 48 h p.i. were 10.6 ± 0.6 and 8.7 ± 0.8, respectively, while uptake of [225Ac]Ac-DOTA-trastuzumab Fab at 18 h p.i. was 3.1 ± 0.5 %ID/g. Elimination from the blood was slowest for [225Ac]Ac-DOTA-trastuzumab IgG followed by F(ab’)2 then Fab. Spleen and liver uptake were greatest for [225Ac]Ac-DOTA-trastuzumab IgG but much lower for F(ab’)2 and Fab. Kidney uptake was highest for [225Ac]Ac-DOTA-trastuzumab Fab. Conclusion: [225Ac]Ac-DOTA-trastuzumab F(ab’)2 provided the highest therapeutic index, inhibiting tumor growth and improving survival while minimizing toxicity. TART with [225Ac]Ac-DOTA-trastuzumab F(ab’)2 is a promising new treatment for HER2-positive BC that could be more effective than trastuzumab. Citation Format: Misaki Kondo, Zhongli Cai, Conrad Chan, Raymond M. Reilly. Optimizing the therapeutic index of targeted α-particle radioimmuotherapy (TART) of HER2-positive breast cancer tumors in NRG mice with 225Ac-labeled trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6030.

Kamin blocking is disrupted by low-dose ketamine in mice: Further implications for aberrant stimulus processing in schizophrenia.

Previous studies have shown that low doses of ketamine, an N-methyl-D-aspartate receptor antagonist, produce aberrantly strong internal representations of associatively activated but absent stimuli in humans and nonhuman animals, suggesting the validity of ketamine treatment as a preclinical model of the positive symptoms of schizophrenia, including hallucinations and delusions. However, whether acute ketamine treatment also impairs the ability to ignore present but informationally redundant stimuli, which is another hallmark of schizophrenia, remains unclear. Accordingly, the present study investigated whether injections of low-dose ketamine attenuate Kamin blocking in an appetitive conditioning preparation in mice. Mice in the blocking group were initially trained with A+ conditioning (i.e., conditioned stimulus A paired with a sucrose solution), followed by compound AX+ training, before the conditioned responses to the cue X were tested in extinction. The animals in the control group received B+ training before the AX+ training. Half of the mice in each group received an injection of 16 mg/kg ketamine before each compound conditioning session and the extinction test, whereas the other half received saline. The results showed a reliable blocking effect in the saline-treated mice, whereas the blocking effect was absent in the ketamine-treated mice. Specifically, the absence of blocking was due to the ketamine-treated mice learning about the blocked cues. This finding further validates the use of low-dose ketamine as a preclinical model of schizophrenia. It also suggests a possible link between hallucination-like aberrant processing of absent events and a reduced ability to suppress attentional processing of task-irrelevant stimuli. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Glutathione supplementation improves fat graft survival by inhibiting ferroptosis via the SLC7A11/GPX4 axis

BackgroundAutologous fat grafting is hampered by unpredictable graft survival, which is potentially regulated by ferroptosis. Glutathione (GSH), a powerful antioxidant used in tissue preservation, has ferroptosis-regulating activity; however, its effects on fat grafts are unclear. This study investigated the effects and mechanisms of GSH in fat graft survival.MethodsHuman lipoaspirates were transplanted subcutaneously into the backs of normal saline-treated (control) or GSH-treated nude mice. Graft survival was evaluated by magnetic resonance imaging and histology. RNA sequencing was performed to identify differentially expressed genes and enriched pathways. GSH activity was evaluated in vitro using an oxygen and glucose deprivation (OGD) model of adipose-derived stem cells.ResultsCompared with control group, GSH induced better outcomes, including superior graft retention, appearance, and histological structures. RNA sequencing suggested enhanced negative regulation of ferroptosis in the GSH-treated grafts, which showed reduced lipid peroxides, better mitochondrial ultrastructure, and SLC7A11/GPX4 axis activation. In vitro, OGD-induced ferroptosis was ameliorated by GSH, which restored cell proliferation, reduced oxidative stress, and upregulated ferroptosis defense factors.ConclusionsOur study confirms that ferroptosis participates in regulating fat graft survival and that GSH exerts a protective effect by inhibiting ferroptosis. GSH-assisted lipotransfer is a promising therapeutic strategy for future clinical application.

Cisplatin and doxorubicin chemotherapy alters gut microbiota in a murine osteosarcoma model.

The gut microbiota is closely associated with tumor progression and treatment in a variety of cancers. However, the alteration of the gut microbiota during the progression and chemotherapy of osteosarcoma remains poorly understood. This study aimed to explore the relationship between dysbiosis in the gut microbiota during osteosarcoma growth and chemotherapy treatment. We used BALB/c nude mice to establish osteosarcoma xenograft tumor models and administered cisplatin (CDDP) or doxorubicin (DOX) intraperitonially once every 2 days for a total of 5 times to establish effective chemotherapy models. Fecal samples were collected and processed for 16S rRNA sequencing to analyze the composition of the gut microbiota. We observed that the abundances of Colidextribacter, Lachnospiraceae_NK4A136_group, Lachnospiraceae_UCG-010, Lachnospiraceae_UCG-006, and Lachnoclostridium decreased, and the abundances of Alloprevotella and Enterorhabdus increased in the osteosarcoma mouse model group compared to those in the control group. In addition, genera, such as Lachnoclostridium and Faecalibacterium were more abundant in chemotherapy-treated mice than those in saline-treated mice. Additionally, we observed that alterations in some genera, including Lachnoclostridium and Colidextribacter in the osteosarcoma animal model group returned to normal after CDDP or DOX treatment. Furthermore, the function of the gut microbiota was inferred through PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), which indicated that metabolism-related microbiota was highly enriched and significantly different in each group. These results indicate correlations between dysbiosis of the gut microbiota and osteosarcoma growth and chemotherapy treatment with CDDP or DOX and may provide novel avenues for the development of potential adjuvant therapies.