Identifying Stress-Exacerbated Thermal-Injury Induced MicroRNAs.

Using a model of combat and operational stress reaction (COSR), our lab recently showed that exposure to an unpredictable combat stress (UPCS) procedure prior to a thermal injury increases pain sensitivity in male rats. Additionally, our lab has recently shown that circulating extracellular vesicle-microRNAs (EV-miRNAs), which normally function to suppress inflammation, were downregulated in a male rat model of neuropathic pain. In this current study, male and female rats exposed to UPCS, followed by thermal injury, were evaluated for changes in circulating EV-miRNAs. Adult female and male Sprague Dawley rats were exposed to a UPCS procedure for either 2 or 4 weeks. Groups consisted of the following: nonstress (NS), stress (S), NS + thermal injury (TI), and S + TI. Mechanical sensitivity was measured, and plasma was collected at baseline, throughout the UPCS exposure, and post-thermal injury. EV-miRNA isolation was performed, followed by small RNA sequencing and subsequent data analysis. UPCS exposure alone resulted in mechanical allodynia in both male and female rats at specific time points. Thermal-injury induction occurring at peak UPCS resulted in increased mechanical allodynia in the injured hind paw compared to thermal injury alone. Differential expression of the EV-miRNAs was observed between the NS and S groups as well as between NS + TI and S + TI groups. Consistent differences in EV-miRNAs are detectable in both COSR as well as during the development of mechanical sensitivity and potentially serve as key regulators, biomarkers, and targets in the treatment of COSR and thermal-injury induced mechanical sensitivity. PERSPECTIVE: This article presents the effects of unpredictable combat stress and thermal injury on EV-contained microRNAs in an animal model. These same mechanisms may exist in clinical patients and could be future prognostic and diagnostic biomarkers.

Exploring combat stress exposure effects on burn pain in a female rodent model.

In the military, constant physiological and psychological stress encountered by Soldiers can lead to development of the combat and operational stress reaction (COSR), which can effect pain management. Similar effects are seen in other populations subjected to high levels of stress. Using a model of COSR, our lab recently showed that four weeks of stress prior to an injury increases pain sensitivity in male rats. With the roles of women in the military expanding and recent studies indicating sex differences in stress and pain processing, this study sought to investigate how different amounts of prior stress exposure affects thermal injury-induced mechanosensitivity in a female rat model of COSR. Adult female Sprague Dawley rats were exposed to the unpredictable combat stress (UPCS) procedure for either 2 or 4 weeks. The UPCS procedure included exposure to one stressor each day for four days. The stressors include: (1) sound stress for 30 min, (2) restraint stress for 4 h, (3) cold stress for 4 h, and (4) forced swim stress for 15 min. The order of stressors was randomized weekly. Mechanical and thermal sensitivity was tested twice weekly. After the UPCS procedure, a sub-set of rats received a thermal injury while under anesthesia. The development of mechanical allodynia and thermal hyperalgesia was examined for 14 days post-burn. UPCS exposure increased mechanosensitivity after two weeks. Interestingly, with more stress exposure, females seemed to habituate to the stress, causing the stress-induced changes in mechanosensitivity to decrease by week three of UPCS. If thermal injury induction occurred during peak stress-induced mechanosensitivity, after two weeks, this resulted in increased mechanical allodynia in the injured hind paw compared to thermal injury alone. This data indicates a susceptibility to increased nociceptive sensitization when injury is sustained at peak stress reactivity. Additionally, this data indicates a sex difference in the timing of peak stress. Post-mortem examination of the prefrontal cortex (PFC) showed altered expression of p-TrkB in 4-week stressed animals given a thermal injury, suggesting a compensatory mechanism. Future work will examine treatment options for preventing stress-induced pain to maintain the effectiveness and readiness of the Warfighter.

Identifying Plasma Derived Extracellular Vesicle (EV) Contained Biomarkers in the Development of Chronic Neuropathic Pain.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n = 6) or sham (n = 6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 postnerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 postnerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include (1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and (2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain. PERSPECTIVE: This article describes the DE miRNA content of plasma derived EVs, comparing neuropathic pain to normal conditions. This data indicates that EV miRNAs may be important in nociception and may also serve as biomarkers for chronic pain. These results encourage further research on EV miRNAs in chronic neuropathic pain sufferers.

Sound-stress-induced altered nociceptive behaviors are associated with increased spinal CRFR2 gene expression in a rat model of burn injury.

Sound stress (SS) elicits behavioral changes, including pain behaviors. However, the neuronal mechanisms underlying SS-induced pain behaviors remain to be explored. The current study examined the effects of SS on nociceptive behaviors and changes in expression of the spinal corticotropin-releasing factor (CRF) system in male Sprague Dawley rats with and without thermal pain. We also studied the effects of SS on plasma corticosterone and fecal output. Rats were exposed to 3 days of SS protocol (n = 12/group). Changes in nociceptive behaviors were assessed using thermal and mechanical pain tests. Following the induction of SS, a subgroup of rats (n = 6/group) was inflicted with thermal injury and on day 14 postburn nociceptive behaviors were reassessed. Spinal CRF receptor mRNA expression was analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). In addition, plasma corticosterone and spinal CRF concentrations were quantified using enzyme-linked immunosorbent assay (ELISA). Increased defecation was observed in SS rats. SS produced transient mechanical allodynia in naive rats, whereas it exacerbated thermal pain in thermally injured rats. Spinal CRFR2 mRNA expression was unaffected by stress or thermal injury alone, but their combined effect significantly increased its expression. SS had no effect on plasma corticosterone and spinal CRF protein in postburn rats. To conclude, SS is capable of exacerbating postburn thermal pain, which is linked to increased CRFR2 gene expression in the spinal cord. Future studies have to delineate whether attenuation of CRFR2 signaling at the spinal level prevents stress-induced exacerbation of burn pain.

Asymmetric drop coalescence launches fungal ballistospores with directionality.

Thousands of fungal species use surface energy to power the launch of their ballistospores. The surface energy is released when a spherical Buller's drop at the spore's hilar appendix merges with a flattened drop on the adaxial side of the spore. The launching mechanism is primarily understood in terms of energetic models, and crucial features such as launching directionality are unexplained. Integrating experiments and simulations, we advance a mechanistic model based on the capillary-inertial coalescence between the Buller's drop and the adaxial drop, a pair that is asymmetric in size, shape and relative position. The asymmetric coalescence is surprisingly effective and robust, producing a launching momentum governed by the Buller's drop and a launching direction along the adaxial plane of the spore. These key functions of momentum generation and directional control are elucidated by numerical simulations, demonstrated on spore-mimicking particles, and corroborated by published ballistospore kinematics. Our work places the morphological and kinematic diversity of ballistospores into a general mechanical framework, and points to a generic catapulting mechanism of colloidal particles with implications for both biology and engineering.

Capillary-inertial colloidal catapults upon drop coalescence.

Surface energy released upon drop coalescence is known to power the self-propelled jumping of liquid droplets on superhydrophobic solid surfaces, and the jumping droplets can additionally carry colloidal payloads toward self-cleaning. Here, we show that drop coalescence on a spherical particle leads to self-propelled launching of the particle from virtually any solid surface. The main prerequisite is an intermediate wettability of the particle, such that the momentum from the capillary-inertial drop coalescence process can be transferred to the particle. By momentum conservation, the launching velocity of the particle-drop complex is proportional to the capillary-inertial velocity based on the drop radius and to the fraction of the liquid mass in the total mass. The capillary-inertial catapult is not only an alternative mechanism for removing colloidal contaminants, but also a useful model system for studying ballistospore launching.

Reliability and validity of digital assessment of perifoveal capillary network measurement using high-resolution imaging.

BACKGROUND: Assessment of the perifoveal capillary network (PCN) might indicate macular function and could reflect the systemic microcirculation. The quantification and reliability of this measurement is currently unknown. The aim of this study was to validate quantification of the PCN by a non-invasive technique from high-resolution retinal images. METHODS: Ten healthy volunteers were included in this validation study. At least 320 high-resolution retinal images were used for assessment of inter- and intra-observer reliability. Non-invasive capillary perfusion mapping was performed using a retinal function imager. After the images were enhanced and segmented, the reproducibility was verified by comparing the values of two independent examiners and of a single examiner at two different time points. RESULTS: The inter-observer concordance coefficients were highly significant for PCN (intraclass correlation coefficient (ICC)=0.901, 95% CI 0.655 to 0.975, p<0.001) and normalised PCN (ICC=0.727, 95% CI 0.262 to 0.923, p=0.004). The intra-observer measurements at two different time points were also highly concordant for PCN (ICC=0.879, 95% CI 0.598 to 0.968, p<0.001) and for normalised PCN (ICC=0.960, 95% CI 0.851 to 0.990, p<0.001). CONCLUSIONS: The reliability of PCN measurement is reproducible and could be used as a new tool to quantify the capillary perfusion network of the macular area.