Adithya Balasubramanian

Pharmacologically induced hypothermia via TRPV1 channel agonism provides neuroprotection following ischemic stroke when initiated 90 min after reperfusion

Traditional methods of therapeutic hypothermia show promise for neuroprotection against cerebral ischemia-reperfusion (I/R), however, with limitations. We examined effectiveness and specificity of pharmacological hypothermia (PH) by transient receptor potential vanilloid 1 (TRPV1) channel agonism in the treatment of focal cerebral I/R. Core temperature (T(core)) was measured after subcutaneous infusion of TRPV1 agonist dihydrocapsaicin (DHC) in conscious C57BL/6 WT and TRPV1 knockout (KO) mice. Acute measurements of heart rate (HR), mean arterial pressure (MAP), and cerebral perfusion were measured before and after DHC treatment. Focal cerebral I/R (1 h ischemia + 24 h reperfusion) was induced by distal middle cerebral artery occlusion. Hypothermia (>8 h) was initiated 90 min after start of reperfusion by DHC infusion (osmotic pump). Neurofunction (behavioral testing) and infarct volume (TTC staining) were measured at 24 h. DHC (1.25 mg/kg) produced a stable drop in T(core) (33°C) in naive and I/R mouse models but not in TRPV1 KO mice. DHC (1.25 mg/kg) had no measurable effect on HR and cerebral perfusion but produced a slight transient drop in MAP (<6 mmHg). In stroke mice, DHC infusion produced hypothermia, decreased infarct volume by 87%, and improved neurofunctional score. The hypothermic and neuroprotective effects of DHC were absent in TRPV1 KO mice or mice maintained normothermic with heat support. PH via TRPV1 agonist appears to be a well-tolerated and effective method for promoting mild hypothermia in the conscious mouse. Furthermore, TRPV1 agonism produces effective hypothermia in I/R mice and significantly improves outcome when initiated 90 min after start of reperfusion.

Role of TRPC1 and TRPC3 Channels in Contraction and Relaxation of Mouse Thoracic Aorta

Background/Aims: Canonical transient receptor potential (TRPC) channels modulate membrane potential and intracellular Ca2+. We examined the role of TRPC1 and TRPC3 channels in vasocontraction and relaxation in mouse aorta. Methods: Vasocontraction and relaxation of aorta from wild-type (WT), TRPC1 KO and TRPC3 knockout (KO) mice were measured for phenylephrine (Phe) and carbachol (CCh). Intracellular Ca2+ was measured in primary aorta endothelial cells (EC) and whole cell K+ current in freshly isolated smooth muscle cells (SMC). Results and Conclusion: TRPC1 KO aorta showed increased vasocontraction to Phe compared to WT and TRPC3 KO aorta due to diminished role of BKCa channels. BKCa mRNA (aorta) and whole cell current (SMC) were reduced versus WT. Contraction in WT aorta was increased to TRPC1 KO level by BKCa channel inhibition. Relaxation to CCh was reduced in TRPC1 KO and TRPC3 KO aortas with concomitant reduction in EC Ca2+ response. Pyr3 (TRPC3 blocker) reduced the Ca2+ response to CCh in EC from WT, but not TRPC3 KO mice. In summary, TRPC1 attenuates receptor-mediated contraction through activation and/or expression of SMC BKCa channels while TRPC3 does not contribute to receptor-mediated constriction. Both TRPC1 and TRPC3 participate in EC Ca2+ influx and vasorelaxation of aorta.

Activation of Endothelial Transient Receptor Potential C3 Channel Is Required for Small Conductance Calcium-Activated Potassium Channel Activation and Sustained Endothelial Hyperpolarization and Vasodilation of Cerebral Artery

Background Transient receptor potential C3 (TRPC3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell (EC) hyperpolarization and endothelium‐dependent hyperpolarization–mediated vasodilation. However, the mechanism by which TRPC3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC3 trafficking to the plasma membrane, where it provides the source of Ca2+ influx for small conductance calcium‐activated K+ (SKCa) channel activation and sustained EC hyperpolarization. Methods and Results Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC3 blocker (Pyr3) produced significant attenuation of endothelium‐dependent hyperpolarization–mediated vasodilation and endothelial Ca2+ response (EC‐specific Ca2+ biosensor) to intraluminal ATP. Pyr3 treatment also resulted in a reduced ATP‐stimulated global Ca2+ and Ca2+ influx in primary cultures of cerebral endothelial cells. Patch‐clamp studies with freshly isolated cerebral ECs demonstrated 2 components of EC hyperpolarization and K+ current activation in response to ATP. The early phase was dependent on intermediate conductance calcium‐activated K+ channel activation, whereas the later sustained phase relied on SKCa channel activation. The SKCa channel–dependent phase was completely blocked with TRPC3 channel inhibition or in ECs of TRPC3 knockout mice and correlated with increased trafficking of TRPC3 (but not SKCa channel) to the plasma membrane. Conclusions We propose that TRPC3 dynamically regulates SKCa channel activation through receptor‐dependent trafficking to the plasma membrane, where it provides the source of Ca2+ influx for sustained SKCa channel activation, EC hyperpolarization, and endothelium‐dependent hyperpolarization–mediated vasodilation.

Shivering and tachycardic responses to external cooling in mice are substantially suppressed by TRPV1 activation but not by TRPM8 inhibition

Mild decrease of core temperature (32-34°C), also known as therapeutic hypothermia, is a highly effective strategy of neuroprotection from ischemia and holds significant promise in the treatment of stroke. However, induction of hypothermia in conscious stroke patients is complicated by cold-defensive responses, such as shivering and tachycardia. Although multiple thermoregulatory responses may be altered by modulators of thermosensitive ion channels, TRPM8 (transient receptor potential melastatin 8) and TRPV1 (TRP vanilloid 1), it is unknown whether these agents affect cold-induced shivering and tachycardia. The current study aimed to determine the effects of TRPM8 inhibition and TRPV1 activation on the shivering and tachycardic responses to external cooling. Conscious mice were treated with TRPM8 inhibitor compound 5 or TRPV1 agonist dihydrocapsaicin (DHC) and exposed to cooling at 10°C. Shivering was measured by electromyography using implanted electrodes in back muscles, tachycardic response by electrocardiography, and core temperature by wireless transmitters in the abdominal cavity. The role of TRPM8 was further determined using TRPM8 KO mice. TRPM8 ablation had no effect on total electromyographic muscle activity (vehicle: 24.0 ± 1.8; compound 5: 23.8 ± 2.0; TRPM8 KO: 19.7 ± 1.9 V·s/min), tachycardia (ΔHR = 124 ± 31; 121 ± 13; 121 ± 31 beats/min) and drop in core temperature (-3.6 ± 0.1; -3.4 ± 0.4; -3.6 ± 0.5°C) during cold exposure. TRPV1 activation substantially suppressed muscle activity (vehicle: 25.6 ± 3.0 vs. DHC: 5.1 ± 2.0 V·s/min), tachycardia (ΔHR = 204 ± 25 vs. 3 ± 35 beats/min) and produced a profound drop in core temperature (-2.2 ± 0.6 vs. -8.9 ± 0.6°C). In conclusion, external cooling-induced shivering and tachycardia are suppressed by TRPV1 activation, but not by TRPM8 inhibition. This suggests that TRPV1 agonists may be combined with external physical cooling to achieve more rapid and effective hypothermia.

Transient receptor potential melastatin 8 channel inhibition potentiates the hypothermic response to transient receptor potential vanilloid 1 activation in the conscious mouse.

Objectives:Mild decrease in core temperature (therapeutic hypothermia) provides lasting neuroprotection following cardiac arrest or cerebral ischemia. However, current methods for producing therapeutic hypothermia trigger a cold-defense response that must be countered by sedatives, muscle paralytics, and mechanical ventilation. We aimed to determine methods for producing hypothermia in the conscious mouse by targeting two transient receptor potential channels involved in thermoregulation, two transient receptor potential (TRP) channels involved in thermoregulation, TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8). Design:Controlled prospective animal study. Setting:Research laboratory at academic medical center. Subjects:Conscious unrestrained young and aged male mice. Interventions:Mice were treated with the TRPV1 agonist dihydrocapsaicin, a TRPM8 inhibitor (“compound 5”), or their combination and the effects on core temperature (Tcore) were measured by implanted thermocouples and wireless transponders. Measurements and Main Results:TRPV1 agonist dihydrocapsaicin produced a dose-dependent (2–4 mg/kg s.c.) drop in Tcore. A loading dose followed by continuous infusion of dihydrocapsaicin produced a rapid and prolonged (> 6 hr) drop of Tcore within the therapeutic range (32–34°C). The hypothermic effect of dihydrocapsaicin was augmented in aged mice and was not desensitized with repeated administration. TRPM8 inhibitor “compound 5” (20 mg/kg s.c.) augmented the drop in core temperature during cold exposure (8°C). When “compound 5” (30 mg/kg) was combined with dihydrocapsaicin (1.25–2.5 mg/kg), the drop in Tcore was amplified and prolonged. Conclusions:Activating warm receptors (TRPV1) produced rapid and lasting hypothermia in young and old mice. Furthermore, hypothermia induced by TRPV1 agonists was potentiated and prolonged by simultaneous inhibition of TRPM8.

MP91-06 INCREASED RISK OF HYPOGONADAL SYMPTOMS IN SHIFT WORKERS WITH SHIFT WORK SLEEP DISORDER

ABSTRACT Objective To examine the association between shift work sleep disorder (SWSD), a primary circadian rhythm disorder characterized by excessive day-time sleepiness associated with shift work, and hypogonadal symptoms in shift workers. Methods Men presenting to an andrology clinic between July 2014 and June 2017 completed questionnaires assessing shift work schedule, SWSD risk, and hypogonadal symptoms ([quantitative] Androgen Deficiency in the Aging Male [qADAM, ADAM]). The impact of nonstandard shift work and SWSD on responses to qADAM and ADAM was assessed using ANOVA and linear regression. Results About 24.1% (619/2571) of men worked nonstandard shifts. Of those, 196 (31.7%) were considered to have SWSD. Controlling for age, comorbidities, and testosterone (T) levels, nonstandard shift workers had qADAM scores 1.12 points lower than day-time workers (P Conclusion Nonstandard shift workers with SWSD have even worse hypogonadal symptoms and lower T levels than day-time workers and nonstandard shift workers without SWSD. This suggests that poor sleep habits, as identified by SWSD, may contribute to the more severe hypogonadal symptoms seen in nonstandard shift workers.

TRPV1-mediated Pharmacological Hypothermia Promotes Improved Functional Recovery Following Ischemic Stroke

Hypothermia shows promise for stroke neuroprotection, but current cooling strategies cause undesirable side effects that limit their clinical applications. Increasing efforts have focused on pharmacological hypothermia as a treatment option for stroke. Previously, we showed that activation of a thermoregulatory ion channel, transient receptor potential vanilloid 1 (TRPV1), by dihydrocapsaicin (DHC) produces reliable hypothermia. In this study, we investigate the effects of TRPV1-mediated hypothermia by DHC on long-term ischemic stroke injury and functional outcome. Hypothermia initiated at 3.5 hours after stroke significantly reduced primary cortical injury. Interestingly, hypothermia by DHC also significantly reduced secondary thalamic injury, as DHC-treated stroke mice exhibited 53% smaller thalamic lesion size. DHC-treated stroke mice further demonstrated decreased neuronal loss and astrogliosis in the thalamus and less thalamic fiber loss by diffusion tensor imaging (DTI). Importantly, a single 8 hour treatment of hypothermia by DHC after stroke provided long-term improvement in functional outcome, as DHC-treated mice exhibited improved behavioral recovery at one month post-stroke. These findings indicate that TRPV1-mediated hypothermia is effective in reducing both primary cortical injury and remote secondary thalamic injury, and a single treatment can produce persistent effects on functional recovery. These data highlight the therapeutic potential for TRPV1 agonism for stroke treatment.

Patient-Specific Virtual Simulation—A State of the Art Approach to Teach Renal Tumor Localization

OBJECTIVE To test whether a novel visuospatial testing platform improves trainee ability to convert two-dimensional to three-dimensional (3D) space. METHODS Medical students were recruited from Baylor College of Medicine and McGovern Medical School (Houston, TX). We 3D reconstructed 3 partial nephrectomy cases using a novel, rapid, and highly accurate edge-detection algorithm. Patient-specific reconstructions were imported into the dV-Trainer (Mimics Technologies, Seattle, WA) as well as used to generate custom 3D printed physical models. Tumor location was altered digitally to generate 9 physical models for each case, 1 with the correct tumor location and 8 with sham locations. Subjects were randomized 1:1 into the dV-Trainer (intervention) and No-dV-Trainer (control) groups. Each subject completed the following steps: (1) visualization of computed-tomographic images, (2) visualization of the reconstructed kidney and tumor in the dV-Trainer (intervention group only), and (3) selection of the correct tumor location on the 3D printed models (primary outcome). Normalized distances from the correct tumor location were quantified and compared between groups. RESULTS A total of 100 subjects were randomized for this study. dV-Trainer use significantly improved subjects ability to localize tumor position (tumor localization score: 0.24 vs 0.38, P < .001). However, subjects in the No-dV-Trainer group more accurately assigned R.E.N.A.L. scores. CONCLUSION Even brief exposure to interactive patient-specific renal tumor models improves a novices ability to localize tumor location. Virtual reality simulation prior to surgery could benefit trainees learning to localize renal masses for minimally invasive partial nephrectomy.

An Infertile Couple's Long and Expensive Path to Varicocele Repairs

OBJECTIVE To characterize the pathways to care that infertile couples requiring varicocele repair (VR) pursue prior to presenting to a male fertility clinic. METHODS An IRB-approved single center retrospective review of patients undergoing VR after presentation to an academic male fertility clinic was performed. Patients whose charts included partner histories were assessed for duration of attempting conception, prior workup, and assisted reproductive technology (ART) use. RESULTS A total of 405 couples were included. At presentation, mean age was 34.4 (SD ± 6.5) years for men and 31.1 (SD ± 4.3) years for women (P < .0001). A couples first visit to a physician was a gynecologist in 59% (198/334) of couples, a reproductive endocrinologist (REI) in 25% (83/334) of cases, with 14% (47/334) presenting without a previous female workup and were self-referred, and 2% (6/334) seeing both a gynecologist and REI prior to presentation. On average, couples attempted pregnancy for 22.3 (SD ± 21.1, range 0-120) months prior to presentation. Eighteen percent of couples underwent ART prior to presentation. Couples who had undergone ART had lower pre-VR total motile sperm counts compared to couples not pursuing ART prior to presentation (P = 0.031). The majority (70.4%) of females had no abnormality in their workup, making varicocele the only correctable factor for infertility in the couple. CONCLUSIONS Our findings show a significant delay in referral of infertile men requiring VR. Eighteen percent of couples underwent costly ART prior to an inexpensive male workup. In an era of medical cost containment, early referral to a male infertility specialist is imperative.

PD46-12 VIRTUAL SIMULATION IMPROVES A NOVICE′S ABILITY TO LOCALIZE RENAL TUMORS IN 3D PHYSICAL MODELS – A MULTI-INSTITUTIONAL PROSPECTIVE RANDOMIZED CONTROLLED STUDY

INTRODUCTION AND OBJECTIVES: Efficient robotic partial nephrectomy requires a precise understanding of tumor location and relationship to vital structures. Translating standard imaging into a reliable 3D mental model is challenging, especially for inexperienced surgeons. We sought to determine if renal tumor visualization and manipulation within a robotic virtual simulator improves the ability of novices to accurately identify tumor location in 3D space. METHODS: We recruited medical students from Baylor College of Medicine and UT McGovern Medical School. Using a custom-built algorithm, two volumetric reconstructions from CT imaging were generated and imported into the dV-Trainer. For each tumor, 9 different model variations were 3D printed (1 real, 8 with modified tumor locations). Subjects were randomized 1:1 into two groups, dV-Trainer and non dV-Trainer, and were given 5 minutes to review CT images. Subjects in the dV-Trainer group were allowed to manipulate the virtual model for an additional 5 minutes. They were then asked to identify the model corresponding to the real tumor in each case and to assign a nephrometry score. The primary outcome was distance of the tumor selected from the correct location. RESULTS: 100 subjects participated and all were included for analysis. There was no difference in subject age (mean: 23.6 2.2) or training year between groups. Subjects in the dV-Trainer group more accurately visualized tumor location (Normalized distance: Model 1: sim 0.17 0.23 vs. no-sim 0.31 0.31, p1⁄40.012; Model 2: sim 0.12 0.28 vs. no-sim 0.34 0.39, p1⁄4.001). These findings were not affected by age or year of training. Surprisingly, subjects in the dV-Trainer group had more difficulty assigning the correct nephrometry score than those in the non-dV-Trainer group. CONCLUSIONS: In this prospective randomized trial, exposure to a patient-specific virtual model improves the novice ability to accurately visualize tumor location when compared to interpreting standard planar CT images alone. This workflow, including our novel reconstruction algorithm, provides a streamlined method for generating patient-specific kidney anatomic simulations which may be valuable for teaching surgical trainees and visualizing complex tumor cases before surgery.