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.

Evaluation of KP-1199: a novel acetaminophen analog for hemostatic function and antinociceptive effects.

BACKGROUND: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples. METHODS: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 µg/ml) and 10× (2140 µg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation. RESULTS: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested. CONCLUSION: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.

Involvement of brain-derived neurotrophic factor (BDNF) in chronic intermittent stress-induced enhanced mechanical allodynia in a rat model of burn pain.

BACKGROUND: Reports show that stressful events before injury exacerbates post-injury pain. The mechanism underlying stress-induced heightened thermal pain is unclear. Here, we examined the effects of chronic intermittent stress (CIS) on nociceptive behaviors and brain-derived nerve growth factor (BDNF) system in the prefrontal cortex (PFC) and hypothalamus of rats with and without thermal injury. RESULTS: Unstressed rats showed transient mechanical allodynia during stress exposure. Stressed rats with thermal injury displayed persistent exacerbated mechanical allodynia (P < 0.001). Increased expression of BDNF mRNA in the PFC (P < 0.05), and elevated TrkB and p-TrkB (P < 0.05) protein levels in the hypothalamus were observed in stressed rats with thermal injury but not in stressed or thermally injured rats alone. Furthermore, administration of CTX-B significantly reduced stress-induced exacerbated mechanical allodynia in thermally injured rats (P < 0.001). CONCLUSION: These results indicate that BDNF-TrkB signaling in PFC and hypothalamus contributes to CIS-induced exacerbated mechanical allodynia in thermal injury state.

Anti-hyperalgesic effects of AG490, a Janus kinase inhibitor, in a rat model of inflammatory pain.

Interleukin 6 (IL-6) has a critical role in pain mechanisms. IL-6 signals through the Janus-activated kinases 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) pathway. The contribution of JAK2 signaling in inflammation-induced hyperalgesia has not been addressed previously. The role of this pathway was investigated using the JAK2 inhibitor, AG490, in a rat model of inflammatory pain. Unilateral hind paw inflammatory pain was induced in male Sprague-Dawley rats by intraplantar (i.pl.) injection of 3.5% ʎ-carrageenan. Inflamed rats received an i.pl. injection of either 3.5% of dimethylsulfoxide or AG490 (1-10 µg). The antinociceptive effects of AG490 were assessed by 2 pain behavioral assays 4 h later: The thermal and mechanical hyperalgesia tests. AG490 (1-10 µg) significantly attenuated ʎ-carrageenan-induced thermal hyperalgesia in a dose-dependent manner. AG490 also reduced mechanical hyperalgesia. Co-administration of opioid receptor antagonist naloxone (10 µg) and AG490 (10 µg) did not reverse AG490-produced antinociceptive activity, suggesting that the µ-opioid receptor is not responsible for the anti-hyperalgesic effects of AG490. Therefore, we suggest that AG490 produces these effects by blocking JAK2 signaling. In conclusion, JAK2 inhibitors may represent a novel class of non-narcotic drugs to treat inflammatory pain.

The intraoperative administration of ketamine to burned U.S. service members does not increase the incidence of post-traumatic stress disorder.

AIM: Patients with severe burns typically undergo multiple surgeries, and ketamine is often used as part of the multimodal anesthetic regimen during such surgeries. The anesthetic ketamine is an N-methyl-D-aspartate receptor antagonist that also provides analgesia at subanesthetic doses, but the psychoactive side effects of ketamine have caused concern about its potential psychological effects on a combat-wounded population. Post-traumatic stress disorder (PTSD) affects approximately 30% of burned U.S. service members injured in Operation Iraqi Freedom/Operation Enduring Freedom. A preliminary analysis by our research group reported that patients who received perioperative ketamine had a significantly lower prevalence of PTSD than those injured service members who did not receive ketamine. We have now expanded this research to examine the relationship between ketamine and PTSD development in a much larger population. METHODS: A retrospective analysis on data from service members being treated for burns at the San Antonio Military Medical Center was conducted. Collected data included drugs received, injury severity score (ISS), total body surface area (TBSA) burned, length of hospital stay (LOS), number of intensive care unit days, number of surgeries, and PTSD Checklist-Military (PCL-M) scores and administration dates. Subjects were grouped based on intraoperative receipt of ketamine, and the groups were compared. The groups were binary for ketamine (yes or no), and dose of ketamine administered was not included in data analyses. Propensity score matching based on ISS and TBSA was performed to control for individual differences in burn severity. RESULTS: Two hundred eighty-nine burned U.S. service members received the PCL-M at least 30 days after injury. Of these subjects, 189 received intraoperative ketamine, and 100 did not. Despite significantly greater injuries, as evidenced by significantly higher TBSA burned and ISS (p < 0.01), patients who received ketamine did not screen positive for PTSD at a different rate than those patients who did not (24% vs. 26.98%, p = 0.582). Patients receiving intraoperative ketamine also underwent a significantly greater number of surgeries, spent more time in the hospital, spent more days in the ICU, and received more morphine equivalent units (p < 0.0001). Propensity score matching based on ISS and TBSA resulted in a total subject number of 130. In the matched samples, subjects who received ketamine still underwent significantly more surgeries and experienced longer hospital stays (p < 0.0001). Again, there was no statistically significant difference in the incidence of a positive screen for PTSD based upon the receipt of ketamine (28% vs. 26.15%, p = 0.843). CONCLUSIONS: Ketamine is often used in burn patients to reduce opioid usage and decrease the hemodynamic and respiratory side effects. Although this study does not show a benefit of ketamine on PTSD development that was identified in previous work with a smaller sample number, it does support the conclusion that ketamine does not increase PTSD development in burned service members.

A rat model of full thickness thermal injury characterized by thermal hyperalgesia, mechanical allodynia, pronociceptive peptide release and tramadol analgesia.

Opioid-related side effects are problematic for burn patients. Dual mechanism therapeutics targeting opioid and non-opioid mechanisms may have reduced side effects with similar analgesic efficacy. Tramadol combines mu opioid receptor agonism with norepinephrine reuptake inhibition and has been effective in treating some types of pain. The effectiveness of tramadol in treating pain associated with burns is unclear. We hypothesized that tramadol is effective in reducing thermal injury-evoked pain behaviors in a rat model. Rats were anesthetized and a 100°C metal probe was placed on the hindpaw for 30 s to induce a full thickness thermal injury. A subset of rats was perfusion fixed and hindpaw tissue and spinal cord collected for anatomical analysis. Rats received morphine (5 mg/kg; i.p.), tramadol (10-30 mg/kg; i.p.) or vehicle and latency to paw withdrawal from a noxious thermal or non-noxious mechanical stimulus was recorded every 10 min over 70 min and again at 2 h. We report that pain behaviors developed within 48 h and peaked at 1 week; paralleled by enhanced expression of pronociceptive neuropeptides in the spinal cord. Morphine and tramadol significantly attenuated hyperalgesia and allodynia, while not significantly altering motor coordination/sedation. These data indicate dual mechanism therapeutics may be effective for treating pain associated with burns.

The relationship between gabapentin and pregabalin and posttraumatic stress disorder in burned servicemembers.

Posttraumatic stress disorder (PTSD) affects approximately 30% of burned Servicemembers returning from Operation Iraqi Freedom/Operation Enduring Freedom. Gabapentin and pregabalin are anticonvulsant drugs that limited evidence suggests may also be effective treatments for some psychological disorders. This study examines the relationship between these anticonvulsants and PTSD development in burned Servicemembers. Drugs received, injury severity score, TBSA burned, length of hospital stay, number of intensive care unit days, number of surgeries, and PTSD Checklist-Military scores and administration dates were collected. Subjects were grouped based on receipt of gabapentin or pregabalin, and the groups were compared. The primary outcome was incidence of a positive screen for PTSD. Because injury severity was significantly different between the two groups, propensity score matching based on injury severity score and TBSA was performed. Two hundred ninety burned Servicemembers received the PTSD Checklist-Military at least 30 days after injury. Of these subjects, 104 received gabapentin, pregabalin, or both and 186 did not. Despite significantly greater injuries, the group that received gabapentin or pregabalin did not develop PTSD at a different rate than those patients who did (P = .727). Propensity score matching resulted in 57 patients in each group; there was no difference between these groups in the incidence of PTSD (P = .663). These data suggest that gabapentin or pregabalin administration may not affect PTSD development in burned Servicemembers. Many factors influence the development and progression of PTSD, but few drugs have been identified that are effective at treating or preventing PTSD.

Relationships between early acute pain scores, autonomic nervous system function, and injury severity in wounded soldiers.

BACKGROUND: Acute pain after injury affects the comfort and function of the wounded soldier and the physiology of multiple body systems. In the civilian population, pain alters the function of the autonomic nervous system, causing increased heart rate and blood pressure. However, there are no data regarding the impact of combat-related pain on physiologic responses. This study is a retrospective analysis that examined the relationship of pain and physiologic parameters in injured soldiers. METHODS: After Institutional Review Board approval, the Joint Trauma Theater Registry (JTTR) was queried to identify soldiers who had pain scores recorded in the Emergency Department (ED) in theater. Subject data collected from the JTTR included the following: pain score, Injury Severity Score (ISS), blood pressure, heart rate, and respiratory rate. RESULTS: We identified 2,646 soldiers with pain scores recorded in the ED. The pain score was not related to most physiologic parameters measured in the ED. Pain intensity had no correlation with blood pressure or heart rate. However, there were relationships between the pain score and respiratory rate, with patients reporting a pain score of 10 having a slightly higher respiratory rate. Increasing pain scores were also associated with increased ISS (p < 0.001). CONCLUSIONS: In contrast to data from civilian patients, early pain scores were not related to heart rate or blood pressure. A pain score of 10 corresponded to an increased respiratory rate. Despite little relationship between pain and injury severity in the civilian population, the increasing ISS was proportional to the pain scale in wounded soldiers.

The relationship of early pain scores and posttraumatic stress disorder in burned soldiers.

Early acute pain after injury has been linked to long-term patient outcomes, including the development of posttraumatic stress disorder (PTSD). Several studies have identified a negative correlation between early anesthetic/analgesic usage and subsequent development of PTSD. This retrospective study examined the relationship between early acute pain and severity of PTSD symptoms in soldiers with burn injuries. Of the soldiers injured in Overseas Contingency Operations who had pain scores recorded at admission to the Emergency Department, 113 had burn injuries. Of those transferred to the military burn center, 47 were screened for PTSD using the PTSD checklist-military (PCL-M) survey at least 1 month after injury. Soldiers with mild, moderate, and severe pain scores had similar Injury Severity Scores and TBSA burned (P = .339 and .570, respectively). However, there were significant differences in PCL-M scores between the mild and severe pain groups (P = .017). The pain levels positively correlated with the PCL-M score (rho = 0.41, P = .004) but not with injury severity markers (Injury Severity Score and TBSA). These data suggest that early acute pain may be related to increased PCL-M score and PTSD symptoms. The intensity of pain was not related to the injury severity, and these data also show no association between pain intensity and physiological measures, including blood pressure and heart rate. However, this is a small sample size, and many other factors likely influence PTSD development. Further study is necessary to explore the relationship between early acute pain and subsequent development of PTSD symptoms.

The relationship of intravenous midazolam and posttraumatic stress disorder development in burned soldiers.

BACKGROUND: Midazolam, a short-acting benzodiazepine, is administered preoperatively and intraoperatively for amnesia and anxiolysis. Subsequently, patients often do not recall events which occurred while they were sedated. Recent studies have also reported retrograde facilitation after midazolam exposure. Posttraumatic stress disorder PTSD is based on memory of a traumatic event. Because of the concern that midazolam may enhance memory of the traumatic event in which soldiers were injured, we investigated the prevalence of PTSD in those burned soldiers who received perioperative midazolam and those who did not. We also investigated the intensity of the memories related to the traumatic event. METHODS: After institutional review board approval, all charts of US soldiers who completed the PTSD Checklist-Military (PCL-M) screening tool (2004-2008) after admission to US Army Institute of Surgical Research were reviewed to determine the number of operations, the anesthetic regime, total body surface area (TBSA) burned, and Injury Severity Score (ISS). RESULTS: The PCL-M was completed by 370 burned soldiers from Operation Iraqi Freedom/Operation Enduring Freedom. During surgery, 142 received midazolam, whereas 69 did not. The prevalence of PTSD was higher in soldiers receiving midazolam as compared with those who did not (29% vs. 25%) (p = 0.481). Both groups had similar injuries based on TBSA and ISS. Patients who received midazolam also had similar scores on PCL-M questions related to memory of the event. CONCLUSIONS: Rates of PTSD are not statistically different in combat casualties receiving midazolam during intraoperative procedures. Intraoperative midazolam is not associated with increased PTSD development or with increased intensity of memory of the traumatic event. Patients receiving midazolam had similar injuries (TBSA and ISS) and underwent a similar number of operations as those not receiving midazolam.

The effect of propranolol on posttraumatic stress disorder in burned service members.

Posttraumatic stress disorder (PTSD) is reported to affect almost one third of the civilian burn patient population. Predisposing factors for PTSD include experiencing a traumatic event. Of Operation Iraqi Freedom/Operation Enduring Freedom (OIF/OEF) soldiers returning home after deployment without injury, 17% reported cognitive symptoms of PTSD. The authors recent study of soldiers burned in OIF/OEF showed a PTSD prevalence of approximately 30%, which is similar to civilian studies. Burns are characterized by hypermetabolism and increased catecholamine levels. beta-Adrenergic receptor blocking agents, like propranolol, decrease catecholamine levels. Propranolol may reduce consolidation of memory and a prophylaxis for PTSD. This retrospective study examines the relationship between PTSD prevalence and propranolol administration. After institutional review board approval, propranolol received, number of surgeries, anesthetic/analgesic regimen, TBSA burned, and injury severity score were collected from patients charts. The military burn center received 603 soldiers injured in OIF/OEF, of which 226 completed the PTSD Checklist-Military. Thirty-one soldiers received propranolol and 34 matched soldiers did not. In propranolol patients, the prevalence of PTSD was 32.3% vs 26.5% in those not receiving propranolol (P = .785). These data suggest propranolol does not decrease PTSD development in burned soldiers. The prevalence of PTSD in patients receiving propranolol is the same as those not receiving propranolol. More research is needed to determine the relationship between PTSD and propranolol.

The correlation between ketamine and posttraumatic stress disorder in burned service members.

BACKGROUND: Predisposing factors for posttraumatic stress disorder (PTSD) include experiencing a traumatic event, threat of injury or death, and untreated pain. Ketamine, an anesthetic, is used at low doses as part of a multimodal anesthetic regimen. However, since ketamine is associated with psychosomatic effects, there is a concern that ketamine may increase the risk of developing PTSD. This study investigated the prevalence of PTSD in Operation Iraqi Freedom/Operation Enduring Freedom (OIF/OEF) service members who were treated for burns in a military treatment center. METHODS: The PTSD Checklist-Military (PCL-M) is a 17-question screening tool for PTSD used by the military. A score of 44 or higher is a positive screen for PTSD. The charts of all OIF/OEF soldiers with burns who completed the PCL-M screening tool (2002-2007) were reviewed to determine the number of surgeries received, the anesthetic regime used, including amounts given, the total body surface area burned, and injury severity score. Morphine equivalent units were calculated using standard dosage conversion factors. RESULTS: The prevalence of PTSD in patients receiving ketamine during their operation(s) was compared with patients not receiving ketamine. Of the 25,000 soldiers injured in OIF/OEF, United States Army Institute of Surgical Research received 603 burned casualties, of which 241 completed the PCL-M. Of those, 147 soldiers underwent at least one operation. Among 119 patients who received ketamine during surgery and 28 who did not; the prevalence of PTSD was 27% (32 of 119) versus 46% (13 of 28), respectively (p = 0.044). CONCLUSIONS: Contrary to expectations, patients receiving perioperative ketamine had a lower prevalence of PTSD than soldiers receiving no ketamine during their surgeries despite having larger burns, higher injury severity score, undergoing more operations, and spending more time in the ICU.