Prepubescent Electronic Cigarette Exposure Affects Sexual Motivation and Puberty in Female But Not Male Long-Evans Rats.

INTRODUCTION: A method for delivering vaporized nicotine to animals has been developed using e-cigarette devices. The present experiment was designed to measure the effects of e-cigarette nicotine on pubertal onset and development of reproductive behavior in female and male Long-Evans rats. AIM AND METHODS: Rats received daily 10-min sessions of electronic-cigarette vaporized nicotine (5% Virginia Tobacco JUUL Pods) or room air in a whole-body exposure chamber (postnatal day 28-31). Pubertal onset was monitored daily (ie, vaginal opening in females, preputial separation in males). Two weeks later, rats were tested for sexual motivation using the partner-preference paradigm, whereby subjects were given the opportunity to approach either a sexual partner or a same-sex social partner. Four weeks later, partner preference was assessed again, 10 min after rats were re-exposed to their same prepubertal treatment. RESULTS: We found that prepubescent electronic-cigarette vaporized nicotine disrupted puberty and sexual motivation in female but not male rats. In vaped females, vaginal opening was delayed and less time was spent with the male stimulus compared to room-air controls. In contrast, no effect of e-cigarette vapor was observed on pubertal onset or on any measures of sexual behavior in male rats. No effects were observed in either female or male rats on the second partner-preference test. CONCLUSIONS: Prepubescent vaporized nicotine affected the development of reproductive physiology and behavior in female rats but not in male rats, whereas an additional acute exposure to nicotine vapor had no effect in either female or male adult rats. IMPLICATIONS: Given the prevalence of increasingly younger users, more animal research is needed to explore the effects of e-cigarette smoking on multiple developmental systems including reproductive physiology and behavior. This model could be useful in exploring multiple behavioral and physiological endpoints in both sexes. Adjustments to the duration of exposure and control conditions will be necessary for future experiments to best model human use.

Updating anatomy and physiology lab delivery: shifting from a paper-based to an online lab instruction platform, just in time for a global pandemic.

This paper describes how an anatomy and physiology laboratory class transitioned from a paper-based lab to an online learning platform that updated the curriculum to rely more on face-to-face small group collaboration and peer teaching. Student perceptions of the new format were positive, but halfway through the transition a global pandemic challenged the new instruction method. The face-to-face curriculum had to be adjusted to a virtual format that lacked in-person interaction between the instructor and the students. This switch to virtual labs had an adverse effect on both student perception and student performance in the second half of the semester. Our observations underscore the importance of creating an interactive community when teaching virtually.

Minocycline blocks glial cell activation and ventilatory acclimatization to hypoxia.

Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation, which persists upon return to normoxia and involves plasticity in both central nervous system respiratory centers and peripheral chemoreceptors. We investigated the role of glial cells in VAH in male Sprague-Dawley rats using minocycline, an antibiotic that inhibits microglia activation and has anti-inflammatory properties, and barometric pressure plethysmography to measure ventilation. Rats received either minocycline (45mg/kg ip daily) or saline beginning 1 day before and during 7 days of chronic hypoxia (CH, PiO2 = 70 Torr). Minocycline had no effect on normoxic control rats or the hypercapnic ventilatory response in CH rats, but minocycline significantly (P < 0.001) decreased ventilation during acute hypoxia in CH rats. However, minocycline administration during only the last 3 days of CH did not reverse VAH. Microglia and astrocyte activation in the nucleus tractus solitarius was quantified from 30 min to 7 days of CH. Microglia showed an active morphology (shorter and fewer branches) after 1 h of hypoxia and returned to the control state (longer filaments and extensive branching) after 4 h of CH. Astrocytes increased glial fibrillary acidic protein antibody immunofluorescent intensity, indicating activation, at both 4 and 24 h of CH. Minocycline had no effect on glia in normoxia but significantly decreased microglia activation at 1 h of CH and astrocyte activation at 24 h of CH. These results support a role for glial cells, providing an early signal for the induction but not maintenance of neural plasticity underlying ventilatory acclimatization to hypoxia.NEW & NOTEWORTHY The signals for neural plasticity in medullary respiratory centers underlying ventilatory acclimatization to chronic hypoxia are unknown. We show that chronic hypoxia activates microglia and subsequently astrocytes. Minocycline, an antibiotic that blocks microglial activation and has anti-inflammatory properties, also blocks astrocyte activation in respiratory centers during chronic hypoxia and ventilatory acclimatization. However, minocycline cannot reverse ventilatory acclimatization after it is established. Hence, glial cells may provide signals that initiate but do not sustain ventilatory acclimatization.

Toll-like receptor signaling adapter proteins govern spread of neuropathic pain and recovery following nerve injury in male mice.

BACKGROUND: Spinal Toll-like receptors (TLRs) and signaling intermediaries have been implicated in persistent pain states. We examined the roles of two major TLR signaling pathways and selected TLRs in a mononeuropathic allodynia. METHODS: L5 spinal nerve ligation (SNL) was performed in wild type (WT, C57BL/6) male and female mice and in male Tlr2-/-Tlr3-/-, Tlr4-/-, Tlr5-/-, Myd88-/-, Triflps2, Myd88/Triflps2, Tnf-/-, and Ifnar1-/- mice. We also examined L5 ligation in Tlr4-/- female mice. We examined tactile allodynia using von Frey hairs. Iba-1 (microglia) and GFAP (astrocytes) were assessed in spinal cords by immunostaining. Tactile thresholds were analyzed by 1- and 2-way ANOVA and the Bonferroni post hoc test was used. RESULTS: In WT male and female mice, SNL lesions resulted in a persistent and robust ipsilateral, tactile allodynia. In males with TLR2, 3, 4, or 5 deficiencies, tactile allodynia was significantly, but incompletely, reversed (approximately 50%) as compared to WT. This effect was not seen in female Tlr4-/- mice. Increases in ipsilateral lumbar Iba-1 and GFAP were seen in mutant and WT mice. Mice deficient in MyD88, or MyD88 and TRIF, showed an approximately 50% reduction in withdrawal thresholds and reduced ipsilateral Iba-1. In contrast, TRIF and interferon receptor null mice developed a profound ipsilateral and contralateral tactile allodynia. In lumbar sections of the spinal cords, we observed a greater increase in Iba-1 immunoreactivity in the TRIF-signaling deficient mice as compared to WT, but no significant increase in GFAP. Removing MyD88 abrogated the contralateral allodynia in the TRIF signaling-deficient mice. Conversely, IFNß, released downstream to TRIF signaling, administered intrathecally, temporarily reversed the tactile allodynia. CONCLUSIONS: These observations suggest a critical role for the MyD88 pathway in initiating neuropathic pain, but a distinct role for the TRIF pathway and interferon in regulating neuropathic pain phenotypes in male mice.

Persistent hyperalgesia in the cisplatin-treated mouse as defined by threshold measures, the conditioned place preference paradigm, and changes in dorsal root ganglia activated transcription factor 3: the effects of gabapentin, ketorolac, and etanercept.

BACKGROUND: Painful neuropathy is a dose-limiting side effect in cancer chemotherapy. To characterize this phenomenon, we examined pain behavior and analgesic actions in a mouse model of cisplatin polyneuropathy. METHODS: Male C57BL/6 mice received intraperitoneal cisplatin or saline (2.3 mg/kg/d) every other day 6 times over 2 weeks for a total dose of 13.8 mg/kg. Thermal escape latencies, mechanical allodynia using von Frey hairs, and observation of behavior/morbidity and body weights were assessed. After onset of allodynia, we examined the actions of intraperitoneal gabapentin (100 mg/kg), etanercept (20 and 40 mg/kg), ketorolac (15 mg/kg), and morphine (1, 3, and 10 mg/kg). Additionally, using the conditioned place preference (CPP) paradigm, we examined the effects of gabapentin and ketorolac on the presumed pain state initiated by cisplatin. Additionally, we examined the spinal cord and dorsal root ganglia (DRG) of cisplatin-treated mice. RESULTS: Cisplatin, but not saline treatment, produced persistent hindpaw tactile allodynia, which persisted 46 days with no effect on thermal escape. Gabapentin and morphine, but neither etanercept nor ketorolac, produced a complete but transient (2-hour) reversal of the allodynia. Etanercept (40 mg/kg) pretreatment resulted in a delay in onset of mechanical allodynia. Using CPP, gabapentin, but not ketorolac, in cisplatin animals resulted in a significant preference for the drug-associated treatment compartment. There was no place preference in non-cisplatin-treated (nonallodynic) mice after gabapentin injection. Immunohistochemistry in cisplatin-treated mice showed no change in glial fibrillary acidic protein (astrocyte) or Iba1 (ionized calcium binding adaptor molecule 1) (microglia) activation states, but a significant increase in activated transcription factor 3 was observed in the DRG. CONCLUSIONS: Cisplatintreated mice display allodynia and an activation of DRG activated transcription factor 3, which is paralleled by its effects on behavior in the CPP system, wherein gabapentin, but not ketorolac, in the presence of the cisplatin polyneuropathy, is positively rewarding, confirming that this neuropathy is an aversive (painful) state that is ameliorated by gabapentin.

A Single 10-Minute E-cigarette Vapor Exposure Reduces Tidal Volume and Minute Ventilation in Normoxia and Normobaric Hypoxia in Adult Rats.

The present study investigated the effects of a single 10-minute exposure to e-cigarette vapor on ventilation in adult male Long-Evans rats. Ventilation was recorded using awake, unrestrained whole-body plethysmography. Baseline recordings were taken the day before full-body exposure to either room air (n = 9; air control group) or e-cigarette vapor (n = 9; treatment group). Post-exposure recordings were taken immediately after the 10-minute room air or vapor exposure. As part of the ventilation protocol, in addition to recording the subject's ventilation in room air, the subjects were also exposed to 10% oxygen (balanced with nitrogen) to assess the effects of e-cigarette vapor on an increased drive to breathe. Ventilation data were analyzed using a 2x2x2 mixed-model ANOVA measuring treatment (vape vs. air) x time (baseline vs. post-treatment) x condition (normoxia vs. hypoxia) for breathing frequency, tidal volume, and minute ventilation. Breathing frequency increased in both treatment groups (air and vape) with exposure to normobaric hypoxia (p < 0.001), with no effect of time (baseline vs. post-treatment) for either group. Tidal volume increased in both treatment groups (air and vape) with exposure to normobaric hypoxia (p < 0.001), and an effect of time (baseline vs. post-treatment) was observed (p = 0.010) for the vape group. Minute ventilation increased in both treatment groups (air and vape) with exposure to normobaric hypoxia (p < 0.001), and an effect of time (baseline vs. post-treatment) was observed (p < 0.001) for the vape group. In conclusion, immediately following a single 10-minute e-cigarette vapor exposure, both tidal volume and minute ventilation were reduced during normoxia and normobaric hypoxia, indicating a decrease in ventilation after a single 10-minute e-cigarette vapor exposure. Furthermore, this exposure also blunted the physiological response to acute hypoxia exposure. Subjects in the vape group, while breathing more rapidly as expected, experienced shallower breathing than the air group during hypoxia. The findings in this study confirm that vaping could result in reduced lung function.

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

The impact of inflammation on respiratory plasticity.

Breathing is a vital homeostatic behavior and must be precisely regulated throughout life. Clinical conditions commonly associated with inflammation, undermine respiratory function may involve plasticity in respiratory control circuits to compensate and maintain adequate ventilation. Alternatively, other clinical conditions may evoke maladaptive plasticity. Yet, we have only recently begun to understand the effects of inflammation on respiratory plasticity. Here, we review some of common models used to investigate the effects of inflammation and discuss the impact of inflammation on nociception, chemosensory plasticity, medullary respiratory centers, motor plasticity in motor neurons and respiratory frequency, and adaptation to high altitude. We provide new data suggesting glial cells contribute to CNS inflammatory gene expression after 24h of sustained hypoxia and inflammation induced by 8h of intermittent hypoxia inhibits long-term facilitation of respiratory frequency. We also discuss how inflammation can have opposite effects on the capacity for plasticity, whereby it is necessary for increases in the hypoxic ventilatory response with sustained hypoxia, but inhibits phrenic long term facilitation after intermittent hypoxia. This review highlights gaps in our knowledge about the effects of inflammation on respiratory control (development, age, and sex differences). In summary, data to date suggest plasticity can be either adaptive or maladaptive and understanding how inflammation alters the respiratory system is crucial for development of better therapeutic interventions to promote breathing and for utilization of plasticity as a clinical treatment.

Toll-like receptor signaling regulates cisplatin-induced mechanical allodynia in mice.

PURPOSE: Cisplatin-treated mice develop a persistent pain state and a condition wherein otherwise innocuous tactile stimuli evoke pain behavior, e.g., tactile allodynia. The allodynia is associated with an up-regulation of activation transcription factor 3 (ATF3) in the dorsal root ganglia (DRG), a factor, which is activated by Toll-like receptors (TLRs). Accordingly, we sought to examine the role of the TLR signaling cascade on allodynia, weight, and changes in DRG ATF3 in cisplatin-treated mice. METHODS: Cisplatin (2.3 mg/kg/day × 6 injections every other day) or vehicle was administered to male wild-type (WT) C57BL/6, Tlr3 (-/-), Tlr4 (-/-), Myd88 (-/-), Trif (lps2) and Myd88/Trif (lps2) mice. We examined allodynia and body weight at intervals over 30 days, when we measured DRG ATF3 by immunostaining. RESULTS: (1) WT cisplatin-treated mice showed tactile allodynia from day 3 through day 30. (2) The Myd88/Trif (lps2) mice did not show allodynia. (3) In Tlr3 (-/-), Tlr4 (-/-), and Myd88 (-/-) mice, withdrawal thresholds were elevated toward normal versus WT cisplatin-treated mice, but remained decreased as compared to vehicle mice. (4) In Trif (lps2) mice, cisplatin allodynia showed a delayed onset, but persisted. (5) In Tlr3 (-/-), Tlr4 (-/-), Myd88 (-/-), and Myd88/Trif (lps2) mice, the increase in DRG ATF3 was abolished. (6) Weight loss occurred during cisplatin administration, which was exacerbated in mutant as compared to WT mice. CONCLUSIONS: Cisplatin evoked a persistent allodynia and DRG ATF3 expression in WT mice, but these effects were reduced in mice with TLR signaling deficiency. TLR signaling may thus be involved in the mechanisms leading to the cisplatin polyneuropathy.

Spinal toll-like receptor signaling and nociceptive processing: regulatory balance between TIRAP and TRIF cascades mediated by TNF and IFNß.

Toll-like receptors (TLRs) play a pivotal role in inflammatory processes, and individual TLRs have been investigated in nociception. We examined overlapping and diverging roles of spinal TLRs and their associated adaptor proteins in nociceptive processing. Intrathecal (IT) TLR2, TLR3, or TLR4 ligands (-L) evoked persistent (7-day) tactile allodynia (TA) that was abolished in respective TLR-deficient mice. Using Tnf(-/-) mice, we found that IT TLR2 and TLR4 TA was tumor necrosis factor (TNF) dependent, whereas TLR3 was TNF-independent. In Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP; Tirap(-/-)) mice (downstream to TLR2 and TLR4), allodynia after IT TLR2-L and TLR4-L was abolished. Unexpectedly, in TIR-domain-containing adapter-inducing interferon-ß (Trif(lps2)) mice (downstream of TLR3 and TLR4), TLR3-L allodynia was abrogated, but intrathecal TLR4-L produced a persistent increase (>21days) in TA. Consistent with a role for interferon (IFN) ß (downstream to TIR-domain-containing adapter-inducing IFNß [TRIF]) in regulating recovery after IT TLR4-L, prolonged allodynia was noted in Ifnar1(-/-) mice. Further, IT IFNß given to Trif(lps2) mice reduced TLR4 allodynia. Hence, spinal TIR domain-containing adaptor protein (TIRAP) and TRIF cascades differentially lead to robust TA by TNF-dependent and independent pathways, whereas activation of TRIF modulated processing through type I IFN receptors. Based on these results, we believe that processes leading to the activation of these spinal TLRs initiate TNF-dependent and -independent cascades, which contribute to the associated persistent pain state. In addition, TRIF pathways are able to modulate the TNF-dependent pain state through IFNß.

Transient tactile allodynia following intrathecal puncture in mouse: contributions of Toll-like receptor signaling.

Studies of spinal drug action in mice often involve percutaneous intrathecal drug administration delivered in a lightly anesthetized animal. A successful lumbar intrathecal (IT) needle stick of a lightly anesthetized (isoflurane) mouse evokes a tail flick, which is an indication of local spinal nerve stimulation. Immediately upon arousal, a hind paw tactile allodynia, as measured with von Frey hairs (pre 1.55±0.11 g vs. injected 0.66±0.08 g) lasts 3-4 h. In a similarly anesthetized mouse without the needle stick, a 1-h allodynia was noted. In studies on spinal Toll-like receptor (TLR) signaling, we observed that following intrathecal puncture and mechanical stimulation of the nerve roots mice deficient in TLR down-stream signaling (Myd88(-/-)/Trif(lps2)), displayed only the transient (1-h) allodynia otherwise observed following isoflurane alone. These data suggest that the extended period of hyperalgesia observed with needle penetration of the dura and mechanical stimulation of the nerve roots requires signaling through the MyD88/TRIF pathways and supports the intrinsic role of Toll-like receptors in the allodynia secondary to the minor nerve activation occurring during the intradural puncture.

Spinal TLR4 mediates the transition to a persistent mechanical hypersensitivity after the resolution of inflammation in serum-transferred arthritis.

Persistent pain after resolution of clinically appreciable signs of arthritis poses a therapeutic challenge, and immunosuppressive therapies do not meet this medical need. To investigate this conversion to persistent pain, we utilized the K/BxN serum transfer arthritis model, which has persistent mechanical hypersensitivity despite the resolution of visible inflammation. Toll-like receptor (TLR) 4 has been implicated as a potential therapeutic target in neuropathic and other pain models. We compared the relative courses of serum transfer arthritis and mechanical hypersensitivity in wild type (WT) and Tlr4(-/-) mice. K/BxN serum transfer induced similar joint swelling and inflammation from days 4-22 in WT and Tlr4(-/-) mice. Unlike WT mice, Tlr4(-/-) mice displayed a significant reversal in mechanical hypersensitivity and diminished appearance of glial activation markers after resolution of peripheral inflammation. Intrathecal (IT) delivery of a TLR4 antagonist, lipopolysaccharide Rhodobacter sphaeroides (LPS-RS; 10 µg), on days 6, 9, and 12 abrogated the transition to persistent mechanical hypersensitivity in WT arthritic mice, while later administration had no impact. We utilized a lipidomics liquid chromatography tandem mass spectrometry methodology to determine spinal cord profiles of bioactive lipid species after early LPS-RS treatment compared to vehicle-treated control animals. WT arthritic mice had reduced spinal levels of the anti-inflammatory prostaglandin 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) on day 6, compared to IT LPS-RS-treated mice. Direct IT application of 15d-PGJ(2) (0.5 µg) on day 6 improved mechanical hypersensitivity in arthritic mice within 15 min. Hence, TLR4 signaling altered spinal bioactive lipid profiles in the serum transfer model and played a critical role in the transition from acute to chronic postinflammatory mechanical hypersensitivity.