Intact Mice Research Articles

BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo.

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3’-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

Dynamics of individual biochemical parameters of blood of intact white mice under the action of the drug “Vitosept”

The active substance of the drug “Vitosept” is high-purity sodium hypochlorite (HPSH), obtained by the direct electrochemical reaction on a newly developed diaphragm-free flow cell, bypassing the formation of molecular chlorine. The developers (Altapharm, Dnipro) used an isotonic (0.9 %) sodium chloride solution prepared in water purified by special technology as the starting electrolyte. The study aimed to determine the effect of different concentrations of Vitosept on the dynamics of serum biochemical parameters of intact white mice. During our research, we found that intragastric administration of even the highest concentrations (500 mg/L) of the drug “Vitosept” did not cause the death of animals or visible signs of intoxication. According to the analysis of clinical manifestations, behavioral reactions, assessment of discomfort, reflex reactions, and the course of metabolic processes, the animals of the experimental groups did not differ from the analogs of the control group. Assessing the protein-synthesizing function of the liver, it was found that the total protein content in the serum of white mice depended on the concentrations of the studied hypochlorite-containing drug and the duration of its receipt. In the animals of the first, second, and third experimental groups during the whole period of the experiment (20 days), the level of total protein in the serum was close to that of the animals of the control group. In animals of groups R4 and R5, on the 10th day of the experiment with the highest applied concentrations of Vitosept (400 and 500 mg/L), the content of total protein in the serum of white mice probably decreased by 8.3 and 7.7 %. It is established that such reduction occurs against the background of changes in the fractional composition of proteins. In white mice of group R5 on the 10th day of the experiment revealed a decrease in blood glucose by 5.9 % (P < 0.05), urea – by 7.4 % (P < 0.05), creatinine – by 9.1 % (P < 0.05). The activity of alkaline phosphatase was 32.1 % (P < 0.05) and GGTP – 70 % (P < 0.05) higher than in the blood of animals of the control group. On the 20th day of the experiment, the level of the studied indicators decreased slightly compared to the similar ones on the 10th day. It was within the limit values, although in most cases, it still differed from the indicators of the animals of the control group. It is obvious that when too high concentrations of even a slightly toxic drug are received, the animal's body reacts with a compensatory-adaptive reaction to the arrival of a foreign substance. Thus, we state that the hypochlorite-containing drug “Vitosept” with the long-term intragastric intake of white mice did not cause hepato- and nephrotoxic effects and revealed some deviations in biochemical parameters in animals of the fourth and fifth groups, on the background of 400–500 mg/L, is most likely the result of adaptive-compensatory response to the intake of the test substance.

RESPONSE OF FIBROBLASTS, MAST CELLS AND LIVER COLLAGEN ON FORMATION OF EXPERIMENTAL AMYLOIDOSIS

The aim of the study was to quantify the response of fibroblasts, mast cells and liver collagen to formation of experimental amyloidosis. Material and methods. All mice were kept on a standard vivarium diet. Experimental mice underwent formation of amyloidosis by subcutaneous administration of soy cream substitute. Intact mice (IG – 3 specimen) and the internal control group for amyloidosis development (KM – 3 specimen) received water in a free access mode. For mice of the second (VM – 3) and third groups (VFM – 3), water was replaced with dry red grape wine "Cabernet Sauvignon" (Crimea) with an ethyl alcohol content of 10–12°, sugar – 5–10 mg/dm³ against the background of amyloidosis. In addition, fructose 5 g/100 ml of wine was added to the wine of VFM group. To assess the relative area of amyloid-free collagen fibers, dewaxed liver sections were stained using the tricolor technique developed by V.A. Kozlov et al. (2017), to assess the relative area of amyloid deposits and to count fibroblasts (FB) – staining with hematoxylin and Congo red, to identify mast cells (TC) and to assess their functionality – Unna's method. The functional state of mast cells was assessed by the degranulation index (ID) and the sulfatation index (IC) which we proposed earlier. The obtained numerical data were used to calculate a new statistical value – the TC/FB index. The data are presented in the form of mean values and median values indicating the percentiles 10 and 90. Differences of the groups were determined using z‑test. Differences in median values were determined using F-test. Results and their discussion. Liver sections of intact mice corresponded to the histological norm. The relative area of amyloid in the liver sections in CM group was 15.2±2.26%, VM group – 1.9±2.1 and VMF group – 2.5±2.9% (p= 0.0000). The relative area of amyloid–free collagen fibers in IG was 0.50±0.18%, in KM is 0.11±0.03 (p = 0.0151), in VM is 0.51±0.16 and in VMF – 0.69±0.18%. ID in IG – 0.38±0.19, KM – 0.39±0.2, VM – 0.55±0.09, VMF – 1.03±0.19 (p = 0.0065 to IG). IS in IG – 0.57±0.19, KM – 0.38±0.2, VM – 0.54±0.09, VMF – 1.23±0.17 (p = 0.0051 to IG). The median values of TC number in IG were 0.8 (0.5¸1.3), KM – 0.4 (0.3¸0.5), VM – 3.0 (2.0¸3.7), VMF – 2.0 (1.3¸3.0), to IG p < 0.05 in all cases. The median values of the FB number in IG were 35.3 (29.9¸39.9), KM – 40.1 (26.1¸55.4), VM – 28.6 (20.3¸33.7), to IG p < 0.05 in all cases, VMF – 51.3 (46.2¸55.4), p > 0.5. The median values of the TC/FB index in IG were 0.022 (0.013¸0.038), KM – 0.009 (0.007¸0.012), p = 0.0000, VM – 0.11 (0.063¸0.167), p = 0.0012, VMF – 0.037 (0.024¸0.06). Conclusions. When forming experimental amyloidosis in the liver of mice, there is a decrease in the number of TC with a parallel increase in the number of FB per field of vision. Substitution of water in the diet of mice by dry red grape wine inhibits the development of changes characteristic of experimental amyloidosis induced by soy cream substitute. A new statistical value – the TC/FB index reflects both the severity of amyloid liver damage and the effectiveness of preventive measures in the form of substituting water by wine.

Abstract 2357: Inhibition of CDK8/19 mediator kinase suppresses primary and metastatic growth of castration-resistant prostate cancer

Abstract Following androgen deprivation therapy (ADT), prostate cancers (PCa) progress to castration resistant prostate cancer (CRPC) status, independent of androgen signaling, through changes in androgen receptor (AR) or by converting to AR-negative forms. Metastatic CRPC remains incurable, and the most potent new drugs prolong survival by only a few months. There is an urgent need for novel agents against advanced CRPC. CDK8 and CDK19 are two isoforms of Mediator kinase, which regulates transcriptional reprogramming, a key process in cancer development, metastasis, and drug resistance. Bioinformatic analysis of clinical prostate cancers shows that CDK19 expression increases during PCa development and progression reaching higher levels than in any other cancers, whereas CDK8 expression increases when PCa becomes CRPC. We have investigated the effects of CDK8/19 Mediator kinase inhibition on gene expression and tumor growth in several CPRC tumor models, growing subcutaneously or in the bone, the principal cause of PCa lethality. CDK8/19 inhibition decreased androgen-stimulated expression of the most strongly androgen-regulated genes, including PSA, in androgen-responsive PCa in vitro and in vivo. CRISPR knockout of both CDK8 and CDK19 in 22Rv1 CRPC cells that express both full-length AR and its androgen-independent V7 variant, had little effect on the tumor growth in intact male mice but strongly suppressed tumor growth in castrated animals. Moreover, re-expression of CDK19 but not of its kinase-dead mutant partially restored tumor growth in castrated mice. Similarly, SNX631, a selective CDK8/19 inhibitor, had little effect on 22Rv1 growth in intact mice but strongly suppressed the growth of these cells (but not of their knockout derivative) in castrated mice. Prolonged (up to 300 days) treatment with SNX631 induced tumor shrinkage and disappearance in a subset of 22Rv1 tumors grown in castrated animals. Transcriptomic analysis revealed that CDK8/19 inhibition or knockout suppressed castration-induced transcriptional reprogramming in tumor cells and affected stromal gene expression. SNX631 also inhibited the growth of a CRPC PDX derived from a PCa patient who failed casodex, abiraterone, and docetaxel, in castrated mice. SNX631 also significantly inhibited in vivo growth of AR-negative PC3 cells both in the flank and in the bone, the principal metastatic site of PCa. These results warrant the development of CDK8/19 inhibitors for the presently incurable metastatic CRPC. Funding acknowledgement: This research was funded by NIH grant R44CA203184 (M.C., I.R., M.L., Y.S.) Citation Format: Jing Li, Thomas Hilimire, Chen Cheng, Eugenia V. Broude, Yueying Liu, Michael B. Lilly, Yusuke Shiozawa, George Wilding, Igor B. Roninson, Mengqian Chen. Inhibition of CDK8/19 mediator kinase suppresses primary and metastatic growth of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2357.

Abstract 3109: Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response

Abstract Background: Preclinical models of prostate cancer are challenging to develop and maintain, especially those that grow in a castrated setting while maintaining receptor and antigen expression. To this end, we have established and characterized a panel of XenoSTART patient-derived (XPDX) prostate models using both intact and castrated athymic nude mice. These models, designated ST1273, ST2347, ST4017, and ST4420, were characterized for receptor expression, genomic alterations, and in vivo drug sensitivity to relevant therapies. Methods: XPDX models representing prostate cancer were established from primary (ST1273) or metastatic (ST2347, ST4017, ST4420) biopsy samples implanted into intact athymic nude mice supplemented with exogenous testosterone. Resulting models were passaged and further developed in both intact and castrated athymic nudes until growth stabilization. Resulting models were characterized using genomic analysis, including WES and RNAseq, receptor expression, and in vivo drug sensitivity studies. Models found sensitive to enzalutamide were conditioned to resistance in vivo by chronic drug administration and resulting models (designated STxxxx/EZR) were characterized and compared with parent lines. For in vivo studies, activity of relevant treatments were benchmarked including enzalutamide administered once daily by oral gavage at 50 mg/kg and docetaxel administered by intravenous injection once weekly at 10 mg/kg. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Each model developed in a castrate or conditioned setting retained similar receptor expression to the parent model including positive AR (2+/3+) and PSMA (2+/3+) staining. Genomic characterization identified a PIK3CA mutation in ST1273 models (PIK3CAE545A), AR mutations in ST2347 (ART878A) and ST4017 (ARH875Y), and a TMPRSS2:ERG fusion in ST4420. In vivo, the ST1273 parent model was found sensitive to enzalutamide (%T/C=11%) but insensitive in castrated mice (%T/C=76%) and the ST1273/EZR model was resistant to enzalutamide in intact (%T/C=56%) or castrated (%T/C=100%) mice. The ST2347 parent model was also found sensitive to enzalutamide (%T/C=19%) but insensitive in castrated mice (%T/C=50%). ST4017 and ST4420 studies in intact and castrated mice are currently underway. All models were sensitive to docetaxel. Conclusion: We have established and characterized a panel of prostate XPDX models using both intact and castrated athymic nude mice and conditioned resistance to enzalutamide by chronic drug administration. These models can be utilized as a valuable tool in better understanding castrate-resistant prostate cancer and in developing novel therapies for enzalutamide-resistant patients. Citation Format: Johnnie Flores, Alyssa Simonson, Dustin Kneifel, Alejandra Diaz, Morgan Harris, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Scott Ulmer, Michael J. Wick. Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3109.

Abstract 2650: Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer

Abstract Background: The androgen receptor (AR), which plays a major role in prostate cancer (PCa), consists of a C-terminal ligand-binding domain (LBD), a hinge domain, a DNA-binding domain (DBD), and an N-terminal domain (NTD). Current AR antagonists and AR signaling inhibitors (ASIs), used in androgen deprivation therapy (ADT) and to prevent disease progression, often fail due to production of alternatively spliced AR variants (AR-Vs) that lack the LBD but retain the DBD, which is essential to AR transcriptional activity. This project examines a series of novel compounds designed to inhibit AR-DNA interaction and study how these compounds affect castration-resistant PCa (CRPC). Methods: Compounds potentially targeting the DBD were used to predict direction and position of compounds when docked to the AR. MTT assays, flow cytometry, immunoblots, qRT-PCR, luciferase assay, and immunofluorescence were used to test compounds on AR and PCa. Drug Affinity Responsive Target Stability (DARTS) tested potential binding of the compounds to the AR. Studies in intact mice were used to determine toxicity of the compounds and the maximum tolerated dose. Results: Of 48 compounds synthesized, C15 had the highest and most consistent efficacy overall. C15 decreased metabolic activity in 22Rv1 and CWR-R1 cells that express AR-Vs and in C4 and C4-2 that do not express AR-Vs. Further, C15 had a reduced effect in AR-null PC3 cells and minimal effect in normal human dermal fibroblast (NHDF) cells. In C4-2 and 22Rv1, C15 significantly reduced PSA mRNA and AR transcriptional activity, and in C4-2, C15 significantly reduced TMPRSS2 and Nrdp1 mRNA levels. C08 and C15 reduced pronase-induced AR proteolysis only when the AR DBD is present. Furthermore, C15 reduces AR binding at the PSA enhancer, but DHT stimulation may weaken this binding inhibition and reduction in AR transcriptional activity. However, combining C15 with an AR inhibitor targeting the LBD (enzalutamide, darolutamide, apalutamide) restores the full inhibitory potential and even reduces metabolic activity and AR transcriptional activity further than either drug alone in both C4-2 and 22Rv1s. No toxicity in intact mice was observed with C15 with the highest dose tested being 50 mg/kg. Conclusion: Our preliminary data demonstrate that C15 directly binds to the DBD of the AR and potently inhibits CRPC cells, including those that express AR-Vs missing the LBD. Although increased DHT levels may weaken C15’s inhibition of the AR DBD, addition of a LBD-targeting AR inhibitor improves the inhibitory potential of both drugs. Based on these results, we conclude that C15 may hold great clinical potential as it successfully inhibits CRPC by targeting the AR DBD with minimal toxicity, and it can improve the effects of standard-of-care LBD-targeting AR inhibitors by reducing AR-V-induced resistance. Citation Format: Thomas M. Steele, Elisabeth A. Messner, Maria Malvina Tsamouri, Salma Siddiqui, Brian Bennion, Ruiwu Liu, Paramita Ghosh. Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2650.

Sex-Dependent Effects of Chronic Microdrive Implantation on Acquisition of Trace Eyeblink Conditioning

Neuroscience techniques, including in vivo recording, have allowed for a great expansion in knowledge; however, this technology may also affect the very phenomena researchers set out to investigate. Including both female and male mice in our associative learning experiments shed light on sex differences on the impact of chronic implantation of tetrodes on learning. While previous research showed intact female mice acquired trace eyeblink conditioning faster than male and ovariectomized females, implantation of chronic microdrive arrays showed sexually dimorphic effects on learning. Microdrive implanted male mice acquired the associative learning paradigm faster than both intact and ovariectomized females. These effects were not due to the weight of the drive alone, as there were no significant sex-differences in learning of animals that received “dummy drive” implants without tetrodes lowered into the brain. Tandem mass tag mass spectrometry and western blot analysis suggest that significant alterations in the MAPK pathway, acute inflammation, and brain derived neurotrophic factor may underlie these observed sex- and surgery-dependent effects on learning.

Aberrant neural activity in prefrontal pyramidal neurons lacking TDP-43 precedes neuron loss.

Mislocalization of TAR DNA binding protein 43 kDa (TARDBP, or TDP-43) is a principal pathological hallmark identified in cases of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As an RNA binding protein, TDP-43 serves in the nuclear compartment to repress non-conserved cryptic exons to ensure the normal transcriptome. Multiple lines of evidence from animal models and human studies support the view that loss of TDP-43 leads to neuron loss, independent of its cytosolic aggregation. However, the underlying pathogenic pathways driven by the loss-of-function mechanism are still poorly defined. We employed a genetic approach to determine the impact of TDP-43 loss in pyramidal neurons of the prefrontal cortex (PFC). Using a custom-built miniscope imaging system, we performed repetitive in vivo calcium imaging from freely behaving mice for up to 7 months. By comparing calcium activity in PFC pyramidal neurons between TDP-43 depleted and TDP-43 intact mice, we demonstrated remarkably increased numbers of pyramidal neurons exhibiting hyperactive calcium activity after short-term TDP-43 depletion, followed by rapid activity declines prior to neuron loss. Our results suggest aberrant neural activity driven by loss of TDP-43 as the pathogenic pathway at early stage in ALS and FTD.

Chronic neurogenic pain inducing chemiluminescence of mitochondrial associates in cardiomyocytes.

e21560 Background: Mitochondrial dysfunction is has a significant impact on the development of heart disease and is a characteristic feature of the heart physiopathology. Our purpose was to analyze some mechanisms of apoptosis regulation and self-organization in the mitochondria of the heart cells in mice with melanoma growing in presence of chronic neurogenic pain (CNP). Methods: The study included female C57BL/6 mice (n = 105) divided into groups: intact animals (n = 21); controls (C, n = 21) with a CNP model created by bilateral sciatic nerve ligation under xylazine/zoletil anesthesia; main group (CNP+B16/F10, n = 63) with B16/F10 melanoma transplanted after CNP creation. After decapitation, mitochondria of the heart were isolated by differential centrifugation. Levels of cytochrome C (ng/mg of protein), caspase 9 (ng/mg of protein), Bcl-2 (ng/mg of protein), AIF (ng/mg of protein), Ca2+ (mmol/g of protein) were determined in mitochondrial samples by ELISA. Results: CNP downregulated levels of Ca2+ by 3.2 times, Bcl-2 by 1.3 times (p < 0.05) and caspase 9 by 1.5 times (p < 0.05), compared to intact mice. AIF levels, on the contrary, were elevated by 2.3 times, and cytochrome C did not differed statistically significantly from intact values. After a week of B16/F10 growth in presence of CNP, levels of Ca2+ in the mitochondria of the heart increased by 5.3 times relative to C values. Further on, Ca2+ decreased to almost undetectable values. The AIF levels changed abruptly: after 1 week it increased by 3.7 times, after 2 weeks it declined to C levels, and after 3 weeks it became 5.2 times lower than in C and 2.3 times lower than in intact animals. Bcl-2 and cytochrome C changed similarly: Bcl-2 after 1 week of melanoma growth in presence of CNP increased 1.7 times (p < 0.05) compared to C, and then after 2-3 weeks it declined and became on average 2.2 times lower than in the mitochondria of the C group; levels of cytochrome C after 1 week did not differed significantly from the values in C, and after 2-3 weeks they decreased by 2.2 times. The levels of caspase 9 in CNP+B16/F10 were on average 2.4 times higher than C values throughout the study. The cold light of high brightness - chemiluminescence was recorded in samples of mitochondrial suspension after 2-3 weeks of CNP+B16/F10. The glow in heart mitochondrial samples was accompanied by bright flashes and a 10-15 second intense white glow with a gradual fading and settling of a large filamentous aggregation of mitochondria on the substrate layer. CNP contributed to the energy supply system blocking in the energy systems of cardiomyocytes. Conclusions: Mitochondrial mechanisms of apoptosis and self-organization of subcellular energy structures in the conditions of malignant tumor growth in presence of CNP are mediated by disruption of polyenzymatic apoptosis regulation systems, and a high level of oxidative stress that induces chemiluminescence of cardiomyocyte mitochondrial associates.

The effects of transplanted adipose-derived multipotent mesenchymal stromal cells from mice of different age or from aging donors in combination with melatonin at experimental parkinsonism

The transplantation of adipose-derived multipotent mesenchymal stromal cells (ADSCs) in Parkinson’s disease/parkinsonism is a promising area in their therapy. The effects of such cells may be influenced by the age of the donor and biologically active factors. The purpose of the study is to compare the effect of transplanted ADSCs of donor mice of different age on the parameters of behaviour, oxidative stress and neuroinflammation in the brain of mice with an experimental model of parkinsonism; to evaluate changes in the effects of cells from older donors under the influence of exogenous hormone melatonin. Materials and methods. The object of the study was adult (5-6 months) and aging (15-17 months) 129/Sv mice. Adult mice were injected once with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and after 17 days – ADSCs of adult or aging donor mice at a dose of 700 thousand cells in the tail vein. Some mice received ADSCs of aging donors in combination with melatonin. Behavioural parameters were assessed in open-field, rigidity and rotarod tests; the relative content of macrophages was measured in the brain, malondialdehyde (MDA), the activity of antioxidant enzymes. Results. Under the influence of MPTP, the number of squares, rearings, body length and length is significantly less than in the intact group, and muscle tone is higher; in the brain the content of MDA and macrophages increases and the activity of superoxide dismutase (SOD) decreases. After the transplantation of adult donor ADSCs, the parameters of body and step length increase significantly, but not to the level of intact mice; the activity of SOD, glutathione reductase (GR) and the proportion of macrophages increase in the brain. After the administration of ADSCs of aging donors, the values of behavioural parameters and the proportion of macrophages in the brain correspond to the control group (only MPTP), and the activity of SOD corresponds to intact animals. In mice treated with ADSCs of aging donors in combination with melatonin, the direction of changes in behavioural parameters, SOD and GR activity, macrophage percentage was similar to that observed after the administration of adult donor ADSCs. Conclusions. The effects of ADSCs transplantation in mice with the MPTP model of parkinsonism depend on the age of the donor and are more pronounced in transplanted cells derived from adult mice. The effects of ADSCs from aging donors in combination with melatonin are consistent with those observed after administration of cells from adult donors.

Olfactory exposure to late-pregnant and lactating mice causes stress-induced analgesia in male mice.

In an attempt to improve reproducibility, more attention is being paid to potential sources of stress in the laboratory environment. Here, we report that the mere proximity of pregnant or lactating female mice causes olfactory-mediated stress-induced analgesia, to a variety of noxious stimuli, in gonadally intact male mice. We show that exposure to volatile compounds released in the urine of pregnant and lactating female mice can themselves produce stress and associated pain inhibition. This phenomenon, a novel form of female-to-male chemosignaling, is mediated by female scent marking of urinary volatiles, such as n-pentyl-acetate, and likely signals potential maternal aggression aimed at defending against infanticide by stranger males.

Behavioral effects and inflammatory markers in the brain and periphery after repeated social defeat stress burdened by Opisthorchis felineus infection in mice

The combination of 4-week repeated social defeat stress (RSDS) and Opisthorchis felineus infection was modeled in C57BL/6 mice. Various parameters were compared between three experimental groups of male mice (SS: mice subjected to RSDS, OF: mice infected with O. felineus, and OF + SS: mice subjected to both adverse factors) and behavior-tested and intact (INT) controls. The combination caused liver hypertrophy and increased the blood level of proinflammatory cytokine interleukin 6 and proteolytic activity of cathepsin B in the hippocampus. Meanwhile, hypertrophy of the spleen and of adrenal glands was noticeable. Anxious behavior in the elevated plus-maze test was predominantly due to the infection, with synergistic effects of an interaction of the two adverse factors on multiple parameters in OF + SS mice. Depression-like behavior in the forced swimming test was caused only by RSDS and was equally pronounced in SS mice and OF + SS mice. Helminths attenuated the activities of cathepsin B in the liver and hypothalamus (which were high in SS mice) and increased cathepsin L activity in the liver. The highest blood level of corticosterone was seen in SS mice but was decreased to control levels by the trematode infection. OF mice had the lowest level of corticosterone, comparable to that in INT mice. Thus, the first data were obtained on the ability of O. felineus helminths—even at the immature stage—to modulate the effects of RSDS, thereby affecting functional connections of the host, namely “helminths → liver↔brain axis.”

Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus.

Hemoglobin (Hb) is the oxygen transport protein in erythrocytes. In blood, Hb is a tetramer consisting of two Hb-alpha (Hb-α) chains and two Hb-beta (Hb-β) chains. A number of studies have also shown that Hb-α is also expressed in neurons in both the rodent and human brain. In the current study, we examined for age-related regulation of neuronal Hb-α and hypoxia in the hippocampus and cerebral cortex of intact male and female mice. In addition, to confirm the role and functions of neuronal Hb-α, we also utilized lentivirus CRISPR interference-based Hb-α knockdown (Hb-α CRISPRi KD) in the non-ischemic and ischemic mouse hippocampus and examined the effect on neuronal oxygenation, as well as induction of hypoxia-inducible factor-1α (HIF-1α) and its downstream pro-apoptotic factors, PUMA and NOXA, and on neuronal survival and neurodegeneration. The results of the study revealed an age-related decrease in neuronal Hb-α levels and correlated increase in hypoxia in the hippocampus and cortex of intact male and female mice. Sex differences were observed with males having higher neuronal Hb-α levels than females in all brain regions at all ages. In vivo Hb-α CRISPRi KD in the mouse hippocampus resulted in increased hypoxia and elevated levels of HIF-1α, PUMA and NOXA in the non-ischemic and ischemic mouse hippocampus, effects that were correlated with a significant decrease in neuronal survival and increased neurodegeneration. As a whole, these findings indicate that neuronal Hb-α decreases with age in mice and has an important role in regulating neuronal oxygenation and neuroprotection.

Neuropilin-1 Identifies a New Subpopulation of TGF-β-Induced Foxp3+ Regulatory T Cells With Potent Suppressive Function and Enhanced Stability During Inflammation.

CD4+Foxp3+ regulatory T cells (Tregs) play a crucial role in preventing autoimmunity and inflammation. There are naturally-derived in the thymus (tTreg), generated extrathymically in the periphery (pTreg), and induced in vitro culture (iTreg) with different characteristics of suppressiveness, stability, and plasticity. There is an abundance of published data on neuropilin-1 (Nrp-1) as a tTreg marker, but little data exist on iTreg. The fidelity of Nrp-1 as a tTreg marker and its role in iTreg remains to be explored. This study found that Nrp-1 was expressed by a subset of Foxp3+CD4+T cells in the central and peripheral lymphoid organs in intact mice, as well as in iTreg. Nrp-1+iTreg and Nrp-1-iTreg were adoptively transferred into a T cell-mediated colitis model to determine their ability to suppress inflammation. Differences in gene expression between Nrp-1+ and Nrp-1-iTreg were analyzed by RNA sequencing. We demonstrated that the Nrp-1+ subset of the iTreg exhibited enhanced suppressive function and stability compared to the Nrp-1- counterpart both in vivo and in vitro, partly depending on IL-10. We found that Nrp-1 is not an exclusive marker of tTreg, however, it is a biomarker identifying a new subset of iTreg with enhanced suppressive function, implicating a potential for Nrp-1+iTreg cell therapy for autoimmune and inflammatory diseases.

Cardiopulmonary protection against nicotine‐induced pulmonary hypertension and right ventricular remodeling in mice is not mediated by ovarian hormones

IntroductionElectronic cigarette use has increased globally prompting a joint committee of international cardiovascular research organizations to call for improved understanding of the health effects of nicotine. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension (PH) and right ventricular (RV) remodeling in male but not female mice, suggesting sex differences in nicotine‐related pathology. Clinically, biological females develop PH more often, but have less severe disease than biological males, likely due to the cardiopulmonary protective effects of estrogen. Nicotine is also metabolized more rapidly in biological females due to sex hormone‐mediated differences in cytochrome P450 activity. These findings led us to hypothesize that female mice are protected against nicotine‐induced PH by a sex hormone‐dependent mechanism. Here, we assessed the role of ovarian hormones in a murine model of nicotine‐induced PH.MethodsFemale C57BL/6J mice were ovariectomized (OVX) one week prior to nicotine exposure. Intact and OVX mice were exposed to nicotine vapor or room air for 12 hours per day for 8 weeks. Nicotine exposure was measured using serum cotinine, which was comparable to human electronic cigarette users, and was not significantly different between intact (421 ± 101 ng/mL) and OVX (314 ± 61 ng/mL) mice. Cardiac structure and function were assessed using echocardiography and RV catheterization.ResultsIn contrast to previous findings in male mice, inhaled nicotine does not cause PH (measured by RV systolic pressure, RVSP) in intact female mice (24.0 ± 0.5 mmHg, n=7 in air vs. 22.7 ± 0.4 mmHg, n=9 in nicotine). RVSP was also unaffected by OVX (24.4 ± 0.9 mmHg, n=9 in air vs. 23.6 ± 0.6 mmHg, n=10 in nicotine). RV free wall thickness during diastole (RV FWT;d) was not significantly different between air‐exposed intact mice (0.328 ± 0.023 mm, n=8), nicotine‐exposed intact mice (0.292 ± 0.015 mm, n=10), air‐exposed OVX mice (0.315 ± 0.021 mm, n=8), and nicotine‐exposed OVX mice (0.293 ± 0.013 mm, n=10). RV internal diameter during diastole (RVID;d) was, likewise, not significantly different between air‐exposed intact mice (1.30 ± 0.03 mm, n=8), nicotine‐exposed intact mice (1.34 ± 0.04 mm, n=10), air‐exposed OVX mice (1.23 ± 0.05 mm, n=8), and nicotine‐exposed OVX mice (1.24 ± 0.05 mm, n=10).ConclusionIn this study, we found no differences in serum cotinine, RVSP, RV FWT;d, or RVID;d between intact and OVX females, leading us to conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine‐induced PH. Due to profound sex differences in clinical PH pathogenesis and nicotine metabolism, further studies (including exploration of androgen‐driven RV remodeling) are necessary to illuminate mechanisms underlying protection from nicotine‐induced pathology in female mice.

Ovariectomy‐Induced Arterial Stiffening is Associated with Downregulation of Tissue Resident Macrophage Markers

Tissue resident macrophages play a major role in the regulation of the extracellular matrix (ECM). The ECM provides an essential scaffold and mechanical stability for blood vessels, but remodeling due to acute injury or chronic inflammatory conditions like hypertension promotes vascular stiffening. We have previously shown that pulse wave velocity (PWV), a measure of arterial stiffness, is higher in male and ovariectomized (OVX) female mice versus intact female mice. RNA‐sequencing of aortas from intact and OVX mice revealed changes in several genes involved in fibrosis as well as macrophage function. Therefore, we hypothesized that estrogen protects from arterial stiffening through maintenance of “protective” macrophages and limiting pro‐fibrotic ECM components. Based on the RNAseq results, we assessed the impact of estrogen on aortic expression of four fibrosis‐associated genes: decorin (DCN), endothelin‐1 (Edn1), type III collagen α1 (Col3a1), and connective tissue growth factor (CTGF) as well as four cell surface macrophage markers: colony stimulating factor 1 receptor (CSF1R), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), AXL receptor tyrosine kinase (Axl), and growth arrest specific 6 (Gas6).MethodsFemale C57BL/6 mice were ovariectomized (OVX) at 8 weeks or remained intact. PWV was obtained as a measure of arterial stiffness using a Vevo 1100 high resolution ultrasound. Mice were euthanized between 20‐23 weeks of age for tissue collection. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using an unpaired t‐test.ResultsAbdominal PWV was increased in OVX mice compared with intact mice (p<0.0001). Surprisingly, OVX induced a significant decrease (p=0.045) in DCN expression, while a downward trend was found for CTGF (p=0.10) and Col3a1 (p=0.08) but not Edn1 (p=0.59). OVX also decreased the expression of macrophage markers CSF1R (p=0.022) and LYVE1 (p=0.047), with a downward trend for Gas6 (p=0.096) but not Axl (0.161).ConclusionIncreased PWV in OVX mice was not accompanied by upregulation of pro‐fibrotic genes but was associated with decreases in macrophage markers CSF1R and LYVE1 in the aorta. Therefore, estrogen loss may promote vascular stiffening through changes in the amounts or functions of specific subsets of macrophages in the vasculature. Our data support previous studies establishing a relationship between CSF1R, LYVE1, and arterial homeostasis. Future studies will examine the impact of estrogen on aortic tissue resident macrophages using flow cytometry and the downstream signaling that promotes arterial stiffening.

Protein kinase G phosphorylation of Chip protects ovariectomized females from myocardial infarction

Proteotoxicity from insufficient clearance of misfolded/damaged proteins exacerbates cardiac functional decline following a myocardial infarction (MI). Estrogen is known to play a crucial role in the survival rate of cardiomyocytes following a MI and is associated with increased protein degradation, however the mechanisms remain to be fully elucidated. Further estrogen has been shown to enhance myocardial protein kinase G (PKG) activity following an insult. In this study, we link this cardioprotective signaling pathway to the carboxyl terminus of Hsc70‐interacting protein (Chip), specifically via phosphorylation at serine 20 (pS20). Chip is a unique effector of proteostasisfunctioning as both a co‐chaperone and ubiquitin ligase. We test the role of Chip S20 with phospho‐mimetic (SE) and phospho‐null (SA) mice compared to WT mice in ovariectomized (OVX) and intact female mice following a MI. We did not observe anydifferences in cardiac function or proteostasis in sham mice regardless of genotype and OVX/intact surgery. Chip SE mice were protected against a MI in both OVX and intact female mice, as exhibited by maintained cardiac function and morphology. Chip WT, even moreso in Chip SA OVX mice, presented with decreased cardiac function, left ventricular chamber dilation and wall thinning, and myocardial fibrosis. Ubiquitinated proteins and protein aggregates were increased in the hearts of Chip SA OVX micewhile Chip SE OVX mice had little accumulation of either, both compared to Chip WT OVX mice. Notably the phospho‐status of Chip S20 did not alter the degradative capacities of the proteasome or lysosome, suggesting the underlying mechanism for enhanced proteostasis is due to increase functionality of the Chip complex. Collectively these dataindicate phosphorylation of Chip S20 is a critical regulator of cardiac proteostasis and is able to afford protection to OVX mice following a MI. These findings have clinical potential as post‐menopausal women are most at risk for deleterious outcomes following a heart attack.

PD07-09 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING

You have accessJournal of UrologyCME1 May 2022PD07-09 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING Srikanth Perike, Calvin VanOpstall, Mathias Morales, Sophia Lamperis, Steve Kregel, Ryan Brown, and Donald J. Vander Griend Srikanth PerikeSrikanth Perike More articles by this author , Calvin VanOpstallCalvin VanOpstall More articles by this author , Mathias MoralesMathias Morales More articles by this author , Sophia LamperisSophia Lamperis More articles by this author , Steve KregelSteve Kregel More articles by this author , Ryan BrownRyan Brown More articles by this author , and Donald J. Vander GriendDonald J. Vander Griend More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is one of the most frequently diagnosed malignancies in men, and its incidence and mortality continue to be a significant clinical problem. Recent evidence from our lab identified MEIS1, an important HOX protein cofactor, as a potential tumor suppressor. Patients bearing MEIS1-positive prostate tumors were significantly less likely to have biochemical recurrence and metastasis compared to men bearing MEIS-negative tumors. Androgen receptor (AR), the major oncogene in PCa, has been shown to interact with HOXB13 to promote prostate cancer progression, but the function of MEIS proteins to antagonize AR/HOXB13 interactions are unknown. We hypothesize that MEIS1 proteins interact with HOXB13 to suppress cancer initiation and progression, and loss of MEIS1 expression in a portion of prostate tumors enables oncogenic AR/HOXB13 interactions. METHODS: We determined the impact of MEIS1 expression and dependency of HOXB13 on AR signaling using cell lines ectopically expressing MEIS1 and/or CRISPR-mediated HOXB13 deletion in both androgen-sensitive LAPC4 and castration-resistant CWR22Rv1 cells. Western blots, qPCR, Proximity Ligation Assay and co-IPs were performed to evaluate the relationship between expression of MEIS1, HOXB13 and AR. Hormonally-intact and castrated male nude mice were used to test the in vivo capability of MEIS1-mediated tumor xenograft formation and rate of tumor growth in the presence and absence of AR ligand. RNA sequencing and informatics analyses was performed on tumor xenografts to determine the global impact of MEIS expression on AR gene targeting in PCa. RESULTS: We found that AR expression was significantly increased when MEIS1 was ectopically expressed compared to controls and HOXB13 knock-out lines. Re-expression of MEIS1 enhanced the binding between HOXB13-MEIS and AR-MEIS, and reduced AR-HOXB13 interaction. In castrated mice, re-expression of MEIS1 significantly decreased the tumor formation and tumor growth rate compared to hormonally intact nude mice. Conversely, xenografts of HOXB13-knockout tumors showed an increased rate of tumor growth and tumor formation compared to MEIS1-expressing cells and controls in both hormonally intact and castrated nude mice. Our preliminary RNA-Seq analysis demonstrate that when MEIS is present, AR pathway activation in response to androgen was associated with increased differentiation and growth suppression. CONCLUSIONS: Our collective data supports our hypothesis that increased MEIS1 expression reduces the AR/HOXB13 interaction and increases the in vivo sensitivity to host castration; this suggests that MEIS-positive cells have decreased oncogenic AR signaling. Future RNA-Seq studies combined with AR ChIP-Seq data will enable us to identify the MEIS-AR targeted genes that are involved in AR pathway modulation that inhibit prostate cancer cell proliferation and suppress tumor progression. These data provide us a strong rationale to support the potential utility of MEIS proteins as predictive clinical biomarkers of metastatic progression. Source of Funding: Funding: This work was supported by the Urology Care Foundation Research Scholar Award sponsored by Chesapeake Urology Associates, DOD grant PC130587, and DOD grant PC180414 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e102 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Srikanth Perike More articles by this author Calvin VanOpstall More articles by this author Mathias Morales More articles by this author Sophia Lamperis More articles by this author Steve Kregel More articles by this author Ryan Brown More articles by this author Donald J. Vander Griend More articles by this author Expand All Advertisement PDF DownloadLoading ...

Kisspeptin Reduces Airway Hyperreactivity and Remodeling in Asthmatic Ovariectomized Mice

RationaleEpidemiological data show changes in asthma occurrence with age and sex. After puberty, increased severity in women shows sex‐skewed occurrence of asthma in females vs. males. This implies the importance of female sex‐steroids, particularly estrogen in asthma pathophysiology. Greater asthma in women may represent detrimental mechanisms upstream to or independent of estrogen. Moreover, data from CNS studies show kisspeptin (Kp) signaling is modulated by estrogen. Our recent in vitro studies show Kp via its receptor, KISS1R regulates airway smooth muscle (ASM) cell proliferation. However, the relevance of endogenous estrogen on Kp/KISS1R signaling and their effect on regulating airway hyperresponsiveness (AHR) and remodeling is not yet explored. Accordingly, in this present study, we utilized intact female vs. ovariectomized (OVX) mice to isolate estrogen influence from the Kp effect in regulating AHR and remodeling in a murine model of allergic asthma.MethodsWild Type C57BL/6J intact female and OVX mice were procured from Jackson Laboratory, USA, and challenged intranasally with a mixed‐allergen (MA) for 28 days. On alternate days, animals from the treatment group received intranasal Kp‐10 (1.2mg/kg), vehicle group received PBS alone. On day 29, mice were anesthetized and subjected to SCIREQ flexiVent to measure lung mechanics like airway resistance (Rrs), elastance (Ers), compliance (Crs), tissue damping (G), and tissue‐elasticity (H). Following this, bronchoalveolar lavage (BAL) was performed for differential leukocyte count (DLC) and lung tissues were processed for histology using hematoxylin and eosin (H&E) and trichrome staining. Additionally, laser‐capture microdissection (LCM) assisted qRT‐PCR analysis in ASM cells was performed for genes relevant to airway remodeling.ResultsMA‐challenged intact female and OVX mice showed a significant increase in Rrs, Ers, G, H, and reduced Crs compared to PBS, more pronounced effects were observed in intact females. Kp‐10 treatment significantly reversed the MA‐induced changes with a decrease in Rrs, Ers, G, H, and increased Crs. Interestingly, in OVX mice we observed significant effects of Kp‐10 to improve the lung mechanics compared to intact females. Notably, Kp‐10 alone exposed group did not show any changes in lung mechanics compared to PBS. DLC in BAL showed Kp‐10 treatment significantly reduced MA‐induced inflammatory cell infiltration. Futhermore, histology studies showed Kp‐10 treatment significantly reduced MA‐induced smooth muscle mass and collagen deposition in airways.ConclusionOverall, this study shows the modulatory role of estrogen in regulating kisspeptin signaling, thereby AHR and remodeling. Thus, providing the relevance to higher asthma incidence and severity in women compared to men.

PD32-07 MICROBIAL EXPOSURE REDUCES SUSCEPTIBILITY TO UROPATHOGENIC E. COLI (UPEC)-INDUCED UROSEPSIS

You have accessJournal of UrologyCME1 May 2022PD32-07 MICROBIAL EXPOSURE REDUCES SUSCEPTIBILITY TO UROPATHOGENIC E. COLI (UPEC)-INDUCED UROSEPSIS Matthew Martin, Whitney Swanson, Tamara Kucaba, Vladimir Badovinac, Molly Ingersoll, and Thomas Griffith Matthew MartinMatthew Martin More articles by this author , Whitney SwansonWhitney Swanson More articles by this author , Tamara KucabaTamara Kucaba More articles by this author , Vladimir BadovinacVladimir Badovinac More articles by this author , Molly IngersollMolly Ingersoll More articles by this author , and Thomas GriffithThomas Griffith More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002583.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary tract infections (UTIs) are the most prevalent infectious disease of the bladder – affecting >130 million people worldwide annually. Uropathogenic E. coli (UPEC) causes most UTIs, starting with bladder colonization and then ascension to the kidneys. In severe cases of pyelonephritis, bacteria can enter the bloodstream, causing urosepsis. The immune system is involved in the development of these pathologies, but little is known about how the basal immune state influences the ability to respond to systemic UPEC infection (urosepsis). Most preclinical studies are conducted with specific pathogen-free (SPF) mice, which possess a largely naïve immune system like neonatal humans. Cohousing (CoH) SPF mice with microbially diverse pet store mice matures the immune system to resemble that of adult humans. Thus, the objective of these studies is to determine how prior microbial exposure affects the immune response to systemic UPEC infection modeling urosepsis. METHODS: For these studies, we infected SPF and CoH mice with UPEC strain UTI89 systemically via intravenous injection. Immune responses, bacterial clearance, and survival following infection was monitored. RESULTS: Interestingly, we found CoH were less susceptible to mortality following systemic infection and cleared UPEC at a faster rate than SPF mice. Following infection, greater numbers of neutrophils, macrophages, CD4 T cells, and antigen-experienced CD8 T cells, and higher amounts of inflammatory cytokines including IFNg were found in the circulation of CoH mice vs. SPF mice. Upon immune cell subset depletion, macrophage-depleted CoH mice showed enhanced mortality to infection similar to SPF mice, and macrophage-depleted CoH mice cleared infection at a slower rate than intact CoH mice, suggesting macrophages are responsible for enhanced protection from systemic UPEC infection in CoH mice. RNA sequencing of macrophages from SPF and CoH mice revealed that microbial exposure alters the macrophage transcriptional profile and suggested cohousing results in a program of innate immunity training. CONCLUSIONS: Our results have important implications in the study of urosepsis, as the data suggest a general history of microbial exposure dramatically alters innate immunity and reduces susceptibility to systemic UPEC infection. While these studies further our understanding of the immune response initiated after systemic UPEC infection, they also suggest mouse studies incorporating exposure to diverse infections may provide a more clinically relevant experimental urosepsis model. Source of Funding: American Urological Association Research Scholar Award (MDM) NIH R21 AI154527-01A1 (TSG) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e549 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Martin More articles by this author Whitney Swanson More articles by this author Tamara Kucaba More articles by this author Vladimir Badovinac More articles by this author Molly Ingersoll More articles by this author Thomas Griffith More articles by this author Expand All Advertisement PDF DownloadLoading ...