ARC25 Agenda – Fri, Jan 31

Phillip Harter1, Omar Sadi Sarkar1, Lewis Chew1, Kevin Goggin2, and Kavitha Yaddanapudi2
1Department of Microbiology & Immunology, , University of Louisville, Louisville, KY, 2Department of Surgery, University of Louisville, Louisville, KY

Introduction/Background. In the United States, lung cancer remains one of the most prevalent and lethal cancers. To improve therapeutic outcomes, much work on understanding the relationship between lymphocytic cells and the tumor microenvironment (TME) has been done, yet there remains a vast gap in knowledge regarding myeloid-derived immune cells and the TME. This study aims to reduce this gap by examining the relationship between CD11b+ Ly6G+ Siglec-F+ neutrophils and the TME. Presently, Siglec-F is most understood as a lectin apoptotic signaling molecule found on the surface of all eosinophils in mice and is analogous to Siglec-8 in humans. However, in recent years, neutrophils have been documented to express Siglec-F and Siglec-8 in several diseased states, including cancer in which they have demonstrated numerous protumor properties. Presently, the mechanisms that drive Siglec-F expression in tumor associated neutrophils and their protumor properties remains unclear, however further exploration could yield potential targets for new immunotherapies.  

Hypothesis/Goal of Study. We hypothesized that Siglec-F neutrophils (SFNs) were induced within the TME rather than the bone marrow (BM) by cytokines produced by the tumor.

Methods and Results. Our study used C57bl-6j mice as a WT control and KRasLSLG12D /+ p53fl/fl as a lung tumor model. Isolated lung and BM SFNs were determined by flow cytometry. BM neutrophils were treated with 10 ng/mL of G-CSF or GM-CSF for 48 hours. B16-F10 cells were modified via a lentivirus system to overexpress GM-CSF/G-CSF and introduced to the lung via IV injection for in vivo confirmation. Additionally, metabolic studies utilizing Mitosox flow staining and Seahorse analysis were done to assess metabolic alterations of SFNs. Statistical analysis was performed in which two-group comparisons were conducted by two-tailed t-tests and multiple comparisons were done by one-way analysis of variance (ANOVA). Statistical significance was determined with a P-value < 0.05.In this study, KRas lung tumor mice displayed a distinct Ly6G+ Siglec-F+ neutrophil population within the lung TME. but these neutrophils were absent in the blood and bone marrow. Our study also identified, via Elisa, marked elevation of G-CSF and GM-CSF produced within the lung tumor microenvironment that, in vitro, displayed the capacity to induce SFNs from purified bone marrow neutrophils. Additionally, our study showed the induction of SFNs by G-CSF/GM-CSF was enhanced, in vitro, by the addition of TGF-β, yet on its own TGF-β was incapable of inducing SFNs. When GM-CSF/G-CSF were over expressed in B16-F10 mice, the neutrophils harvested from the modified B16-F10 cells saw a significant increase in Siglec-F expression compared to unmodified B16-F10 which express little GM-CSF/G-CSF. Finally, in comparison to Siglec-F- neutrophils, SFNs displayed increased total ROS, mtROS, and mitochondrial potential suggesting these neutrophils exhibit an alerted metabolic state.

Discussion/Conclusions. In summary, our data suggests that SFNs are a distinct subpopulation of LyG+ neutrophils generated in a tumor-dependent manner contingent upon the presence of G-CSF/GM-CSF produced within the TME, and the effects are enhanced by the expression of TGF-β. Our data suggest that SFNs are more metabolically active than SiglecF- neutrophils though the effect of this finding on the development of the tumor has yet to be determined. Due to the well-documented capability of tumors to convert myeloid cells into immune suppressive cells marked by increased ROS levels, further examining the relationship between SFNs and T-cell activity warrants further investigation.

Citation/Acknowledgements.  This work was supported in part by grant 5P20GM103436-22 (KY INBRE) and grant 5P20GM135004-03 (CCII COBRE). Additionally grant support was provided by the NIH.

Sujata Saraswat Ohri, Scott A. Myers, Benjamin Rood, Brandon L. Brown, Paula M. Chilton, Lukasz Slomnicki, Yu Li, George Z. Wei, Kariena R. Andres, Russell M. Howard, Scott R. Whittemore, and Michal Hetman

Kentucky Spinal Cord Injury Research Center and Department of Neurological Surgery, University of Louisville, School of Medicine, Louisville, KY

Introduction/Background. Cellular stressors inhibit general protein synthesis while upregulating stress response transcripts and/or proteins. Phosphorylation of the translation factor eIF2α by one of the several stress-activated kinases is a trigger for such signaling, known as the integrated stress response (ISR). The ISR regulates cell survival and function under stress.  

Hypothesis/Goal of Study. ISR activation is documented after traumatic spinal cord injury (SCI). Moreover, germline deficiency of the downstream ISR transcription factor CHOP/DDIT3 reduces SCI-associated tissue damage and improves functional recovery. However, it is unknown which eIF2α kinases that initiate ISR contribute to SCI pathogenesis. Therefore, post-SCI tissue damage and functional recovery were analyzed in germline knockout mice of three major ISR kinases Eif2ak1/Hri, Eif2ak2/Pkr, and Eif2ak4/Gcn2.

Methods and Results. One-day post-injury (moderate contusive SCI at the T9 level), reduced levels of peIF2α were found in Hri-/- and Gcn2-/-, but not in Pkr-/- mice. In addition, Hri-/- mice showed attenuated expression of the downstream ISR transcripts, Atf4 or Chop. Such differential effects of SCI-activated ISR correlated with a strong or moderate enhancement of locomotor recovery in Hri-/- or Gcn2-/- mice, respectively. Hri-/- mice also showed reduced white matter loss, increased content of oligodendrocytes and attenuated neuroinflammation including decreased lipid accumulation in microglia/macrophages. Cultured neonatal Hri-/- oligodendrocytes showed lower ISR cytotoxicity. Moreover, cell-autonomous reduction of neuroinflammatory potential was observed in microglia and bone marrow-derived macrophages derived from Hri-/- mice.

Discussion/Conclusions. These data identify HRI as a major positive regulator of SCI-associated white matter damage. In addition, targeting HRI may enable multimodal neuroprotection to enhance functional recovery after SCI. Mechanistic insight into the role of HRI in SCI-induced neuroinflammation will be obtained by the ongoing transcriptomic analysis of SCI tissue from wt vs. Hri-/- mice.

Citation/Acknowledgements.  NS073584, NS103433, NS108529, and  P20GM103436-24 (KY INBRE Voucher Program) 

I. Mark Olfert1, Eiman Aboaziza2, Lainey Shouldis3, Li Ma3, Amber Mills1, and Gangqing Hu3

1Department of Physiology, Pharmacology & Toxicology, Division of Exercise Physiology and Center for Inhalation Toxicology, West Virginia University School of Medicine, Morgantown, WV, 2National Institute on Drug Abuse, National Institutes of Health, Bethesda, MD, Axle Informatics, Rockville, MD, 3 Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, WV

Abstract Embargoed

Sara Palacio1, James Campbell1, Fang Fang Xu1, Allan Butterfield2, Subbarao Bondada3, Heidi Weiss4, John Villano5, William St. Clair6, Daret St. Clair1, and Luksana Chaiswing1

1Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY, 2Department of Chemistry, University of Kentucky, Lexington, KY, 3Department of Microbiology, University of Kentucky, Immunology and Molecular Genetics, Lexington, KY, 4Biostatistics and Bioinformatics, University of Kentucky, Lexington, KY, 5Department of Neuro-Oncology, University of Kentucky, Lexington, KY, 6Department of Radiation Oncology, University of Kentucky, Lexington, KY

Introduction/Background. Glioblastoma (GBM) is among the most aggressive and fatal cancers, with a median survival of only 15–24 months. Novel therapeutic approaches are urgently needed, but their development is hindered by a lack of sensitive methods to detect unique GBM features. Extracellular vesicles (EVs) are promising as liquid biopsy platforms because they are released by all cells and reflect the molecular characteristics of their tissue of origin. GBM cells generate high levels of reactive oxygen species (ROS), producing the toxic lipid peroxidation product 4-hydroxynonenal (4HNE), which modifies proteins. Our research identified that GBM cells release EVs enriched in 4HNE-bound proteins (RedoxEVs). However, the localization of 4HNE-bound proteins in EVs and their biological role in GBM pathology remain unclear.  

Hypothesis/Goal of Study. We hypothesize that GBM-derived RedoxEVs activate microglia, inducing further ROS release and neurotoxicity. Our goal is to localize 4HNE-bound proteins on or within RedoxEVs using advanced imaging techniques, addressing limitations in conventional detection methods.

Methods and Results. We propose using the FEI Talos F200X transmission electron microscope (TEM) with immunogold staining to visualize 4HNE-bound proteins in RedoxEVs. This method will identify whether these proteins are present on the EV membrane, inside as cargo, or both. Preliminary results suggest RedoxEVs are enriched in 4HNE-bound proteins, supporting their potential role in GBM-induced neurotoxicity.

Discussion/Conclusions. This study aims to establish a proof-of-concept method for RedoxEV detection using immunogold labeling coupled with advanced TEM imaging. If successful, it will provide critical insights into the role of RedoxEVs in GBM pathophysiology and offer a powerful tool for their detection, enabling improved diagnostics and therapeutic strategies for GBM patients.

Citation/Acknowledgements. This project was supported by NIH/NIGMS grant P20GM103436-24 (KY INBRE), P20GM148326 (UKY COBRE-CNS Metabolism), and P20GM121327 (UKY COBRE-CCM).

Aastha Suthar1,2, Andrea Willhite1, Kristin Benton1, Aaron Bullock1, Siqi Wang1, and Alexander Ovechkin1,2

1Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, KY, 2Department of Neurological Surgery, University of Louisville, Louisville, KY

Introduction/Background. Respiratory and cardiovascular dysfunction, including systemic and orthostatic hypotension, is a significant source of disability in individuals with chronic spinal cord injury (SCI). Despite its prevalence, no effective rehabilitative strategies are currently available to stabilize blood pressure in this population. Our previous studies demonstrated that respiratory training (RT) enhances pulmonary function and improves baroreflex sensitivity in individuals with SCI. We also showed that neuromodulatory lumbosacral spinal cord epidural stimulation (scES), configured to target autonomic responses, acutely increases arterial blood pressure in these patients.  

Objective/Hypothesis. This study aimed to evaluate the impact of neuromodulatory rehabilitation on baroreflex sensitivity (BRS) as a step toward developing evidence-based rehabilitation strategies for patients with SCI. We hypothesized that combining RT with scES, specifically configured to activate sympathetic networks, would produce a synergistic increase in BRS in individuals with chronic SCI.

Methods and Results. Methods: Six participants with cervical SCI were divided into three groups (two participants per group). Each group underwent 80 sessions of the following interventions: Group 1: Respiratory training (RT); Group 2: spinal cord Epidural Stimulation (scES); Group 3: Combined RT+scES. BRS was assessed before and after the interventions by analyzing beat-to-beat systolic blood pressure plotted against electrocardiographic R-R intervals using a linear regression. Sympathetic activity was characterized by evaluating slopes during Phase IV of the Valsalva maneuver and the 5-second maximum expiratory pressure (5-s PEmax) tests. Inter-group comparisons were performed to identify differences in outcomes.

Results: Post-intervention, BRS improved in five out of six participants during both the Valsalva and 5-s PEmax maneuvers, with significant increase observed in Group 3.

Conclusion. These findings suggest that neuromodulatory rehabilitative strategies combining RT with scES can improve hemodynamic responses in individuals with respiratory-cardiovascular complications of chronic SCI. This combined approach shows promise as a novel therapeutic option.

Citation/Acknowledgements. This research was supported by the National Institutes of Health Heart, Lung, and Blood Institute (R01 HL150581), University of Louisville Hospital, and Medtronic PLC.

Mosammat Perveen, Matthew Biernat, Jessica Sackett, and Sujin Bao

Kentucky College of Osteopathic Medicine, University of Pikeville, Pikeville, KY

Introduction/Background. The formation of memory and its consolidation into a neural footprint, commonly referred to as an engram, involves the interplay of various cortical and subcortical structures. Contextual fear conditioning (CFC), a widely used paradigm for studying memory, has historically highlighted the hippocampus and amygdala as key facilitators of contextual memory formation, consolidation, and retrieval. Recent studies, however, underscore the roles of additional regions, including the prefrontal cortex, retrosplenial cortex, and thalamus, within this complex neural network. Despite significant progress, integrating these structures into a cohesive framework remains a challenge as researchers strive to characterize their respective roles and contributions to the neural map of memory. 

Hypothesis/Goal of Study. In this review, we aim to consolidate recent advances in the neural circuitry underlying contextual memory and introduce an integrated model of contextual fear memory.

Methods and Results. We conducted a literature search. 

Databases used: PubMed, ScienceDirect, Scopus, and Web of Science.  

Keywords used: learning and memory, neural circuitry, memory acquisition, consolidation, retrieval, rodent, contextual fear conditioning

Inclusion criteria: research article

Exclusion criteria: review, editorial, news

Results: We found 17 research articles. 

These articles were divided into 3 groups: 

1. 1. Studies that concern memory acquisition (6)

   2. Studies that address memory consolidation (3)

   3. Studies that concern memory retrieval (8)

Our analyses highlight that five brain regions play critical roles in the development of contextual fear memory: the amygdala, hippocampus, prefrontal cortex, retrosplenial cortex, and postrhinal cortex.

Discussion/Conclusions. Each brain region plays a unique but integrative role in the acquisition, consolidation, and retrieval of contextual fear memory.  Specifically, the hippocampus is absolutely required for memory acquisition while the prefrontal cortex is essential for the consolidation, reconsolidation, and retrieval of long-term memories. In rodents, lesions in the prefrontal cortex result in impaired long-term social memory, while short-term social memory remains unaffected. Our analyses of the literature also highlight that the retrosplenial cortex contains a parallel neural circuit that also plays an essential role in memory acquisition, systemic consolidation, and retrieval.

Citation/Acknowledgements. Funding support: KY INBRE Student Diversity Research Award (SDRA) Subaward # ULRF_23-1033-10

Mikki Rodgers, Mackenzie Feltner, Asia Good, Taylor Easybuck, Alexandria Easton, and Christine Perdan Curran

Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY

Introduction/Background. Pollutants that bind the aryl hydrocarbon receptor (AHR) are frequently associated with changes in monoamine neurotransmitter levels. These include polycyclic aromatic hydrocarbons (PAHs) and the prototypical PAH benzo[a]pyrene. Human studies have found a strong correlation between early life exposure to PAHs and persistent effects on cognition and behavior. To identify those at highest risk, we use a mouse model with genetic variations in the AHR pathway. In the studies presented here, we focus on changes in neurotransmitter levels in key brain regions required for learning and memory and motor function. 

Objective/Hypothesis. We hypothesize that the greatest changes will be seen in mice with genetic differences that make them less capable of metabolize and clearing BaP.

Methods and Results. We treated wild type Cyp1a2(+/+) and knockout Cyp1a2(-/-) mice from gestational day 10 until their pups were weaned at postnatal day 25 (P25). We conducted a battery of neurobehavioral tests starting at P60 and collected striatum, hippocampus and prefrontal cortex following behavior around P120. We used high-performance liquid chromatography (HPLC) with electrochemical detection to quantify dopamine, serotonin and their metabolites. We found no significant differences in the striatum, but multiple differences in the hippocampus and prefrontal cortex. There was a trend for a gene x treatment interaction in the hippocampus with BaP-exposed wild type mice having lower levels whereas BaP exposed knockouts had higher levels compared with controls (P = 0.083). In the prefrontal cortex, BaP-treated mice had higher levels of dopamine (P = 0.053), but no differences in the metabolite DOPAC. There was also a significant gene x treatment interaction with higher levels of serotonin in the wild type mice and lower levels in the knockouts (P = 0.01).

Discussion/Conclusions. Our data suggest that developmental BaP exposure has the greatest effect on monoamine neurotransmitters in brain regions associated with cognitive function.

Citation/Acknowledgements. This project was supported by grants P20GM103436-24 (KY INBRE) from the National Institute of General Medical Sciences, and R15ES020053, R15ES030541 and R03ES035480 from the National Institute of Environmental Health Sciences (Curran).

Jaime Heckler1, Lily Faulkenberg1, Carly McPherson1, Camille Ratcliff1, Isaiah Hisler1, Carmelo Venero2, Gordon Baylis1, Alex Morgan3, and Patrick Ledwidge1

1Department of Psychological Sciences, Western Kentucky University, Bowling Green, KY, 2College of Medicine, University of Kentucky, Bowling Green, KY, 3Department of Allied Health, Sport and Wellness, Baldwin Wallace University, Berea, OH

Introduction/Background. Understanding how cognitive and motor systems interact is important for interpreting an individual’s accuracy in performing run-to-cut sidestep movements, especially when movements are complex or call for rapid decision-making, such as in sports like football and soccer. This study examined how factors such as direction anticipation, congruence, and limb dominance may influence one’s ability to execute these movements. 

Hypothesis/Goal of Study. Accuracy in performing a sidestep cut will be lower when cut direction is unanticipated than anticipated, particularly for incongruent flanker stimuli. Accuracy will be lower when planting on non-dominant limb (i.e. right cuts) irrespective of anticipation.

Methods and Results. Methods: Eighteen right-limb dominant athletes completed 192 trials of a run-to-cut maneuver in which they jogged and performed a sidestep change of direction (“cut”) at the end of a 12-foot runway. A flanker stimulus appeared during the run phase. The middle arrow was congruent or incongruent with the other arrows and indicated a left (< < < < < or > > < > >) or right cut (> > > > > or < < > < <). A pre-cue was presented before 50% of trials to manipulate the anticipation of the cut direction (unanticipated v. anticipated). A correct trial was of the appropriate speed (4.05-4.95 mph), cut direction, without gait errors, and the plant limb was contralateral to cut direction.

Results: Accuracy did not significantly vary between anticipated and unanticipated conditions, and there was no interaction between congruence and anticipation (p’s > .05). Accuracy was lower when participants planted on their nondominant limb (right cut) than dominant limb (left cut), F(1,17) = 13.41, p = .002. There was not a significant interaction between limb dominance and anticipation (p > .05).

Discussion/Conclusions. Rapid sidestep cutting maneuvers are more challenging when planting on the non-dominant limb, irrespective of direction anticipation and stimulus congruence.

Citation/Acknowledgements. KY INBRE (NIGMS grant # 8P20GM103436); WKU Office of Research and Creative Activity.