Vancomycin modestly attenuates symptom severity during onset of and recovery from exertional heat stroke in mice.

Increased intestinal permeability during exertion and subsequent leakage of bacteria into circulation is hypothesized to accelerate exertional heat stroke (EHS) onset and/or exacerbate EHS severity. To provide proof of concept for this theory, we targeted intestinal microbiota via antibiotic prophylaxis and determined whether vancomycin would delay EHS onset and/or mitigate EHS severity and mortality rates using a mouse model of EHS. Mice were 1) designated as EHS or Exercise Control (ExC) and 2) given 7 days of vancomycin (VEHS, VExC) or untreated water (EHS, ExC) before EHS/Exercise. Following EHS/ExC, mice were euthanized immediately (0 h) or returned to their home cage (25°C) and euthanized after 3 h or 24 h. VEHS mice exhibited reduced abundance and altered composition of fecal bacteria (with notable decreases in genera within orders Clostridiales and Bacteroidales); increased water consumption, lower core temperature (TC) before and during heating (TCMax), lower circulating markers of organ damage and inflammation at 24 h; and reduced hepatic activation of stress pathways at 0 and 3 h compared with EHS mice. Vancomycin-induced alterations to the intestinal microbiota likely influenced EHS outcomes, but it is unconfirmed whether this is due to attenuated bacterial leakage into circulation or other (in)direct effects on physiology and behavior (e.g., decreased TC, increased water consumption). To our knowledge, this is the first study quantitating antibiotic effects in conscious/unanesthetized, exertional HS animals.NEW & NOTEWORTHY Vancomycin prophylaxis lowered core temperature before and during EHS, mitigated EHS-associated rise of hepatic biomarkers and cytokines/chemokines in circulation (particularly at 24 h), and corresponded to inhibited phosphorylation of hepatic c-Jun NH2-terminal kinase on Threonine 183/Tyrosine 185 at 0 and 3 h in conscious, unanesthetized mice. However, vancomycin also induced cecal enlargement suggesting its off-target effects could limit its utility against EHS.

Amino acid solution mitigates hypothermia response and intestinal damage following exertional heat stroke in male mice.

Increased gut permeability is implicated in the initiation and extent of the cytokine inflammatory response associated with exertional heat stroke (EHS). The primary objective of this study was to determine if a five amino acid oral rehydration solution (5AAS), specifically designed for the protection of the gastrointestinal lining, would prolong time to EHS, maintain gut function and dampen the systemic inflammatory response (SIR) measured during EHS recovery. Male C57/BL6J mice instrumented with radiotelemetry were gavaged with 150 µL of 5AAS or H2 O, and ≈12 h later were either exposed to an EHS protocol where mice exercised in a 37.5°C environmental chamber to a self-limiting maximum core temperature (Tc,max) or performed the exercise control (EXC) protocol (25°C). 5AAS pretreatment attenuated hypothermia depth and length (p < 0.005), which are indicators of EHS severity during recovery, without any effect on physical performance or thermoregulatory responses in the heat as determined by percent body weight lost (≈9%), max speed (≈6 m/min), distance (≈700 m), time to Tc,max (≈160 min), thermal area (≈550°C∙min), and Tc,max (42.2°C). EHS groups treated with 5AAS showed a significant decrease in gut transepithelial conductance, decreased paracellular permeability, increased villus height, increased electrolyte absorption and changes in tight junction protein expression pattern suggestive of improved barrier integrity (p < 0.05). No differences were witnessed between EHS groups in acute phase response markers of liver, circulating SIR markers, or indicators of organ damage during recovery. These results suggest that a 5AAS improves Tc regulation during EHS recovery through maintaining mucosal function and integrity.

Identification of therapeutic targets in a murine model of severe exertional heat stroke.

Exertional heat stroke (EHS) is a potentially lethal condition resulting from high core body temperatures (TC) in combination with a systemic inflammatory response syndrome (SIRS) with varying degrees of severity across victims, and limited understanding of the underlying mechanism(s). We established a mouse model of severe EHS to identify mechanisms of hyperthermia/inflammation that may be responsible for organ damage. Mice were forced to run on a motorized wheel in a 37.5°C chamber until loss of consciousness and were either removed immediately (exertional heat injury or EHI; TCMax = 42.4 ± 0.2°C) or remained in the chamber an additional 20 min (EHS; TCMax = 42.5 ± 0.4°C). Exercise control mice (ExC) experienced identical procedures to EHS at 25°C. At 3 h post-EHS, there was evidence for an immune/inflammatory response as elevated blood chemokine [interferon γ-induced protein 10 (IP-10), keratinocytes-derived chemokine (KC), macrophage inflammatory proteins (MIP-1α), MIP-1ß, MIP-2] and cytokine [granulocyte colony-stimulating factor (G-CSF), interleukins (IL-10), IL-6] levels peaked and were highest in EHS mice compared with EHI and ExC mice. Immunoblotting of organs susceptible to EHS damage indicated that several kinases were sensitive to stress associated with heat/inflammation and exercise; specifically, phosphorylation of liver c-Jun NH2-terminal kinase (JNK) at threonine 183/tyrosine 185 immediately (0 h) postheating related to heat illness severity. We have established a mouse EHS model, and JNK [or its downstream target(s)] could underlie EHS symptomatology, allowing the identification of molecular pathways or countermeasure targets to mitigate heat illness severity, enable complete recovery, and decrease overall EHS-related fatalities.

Impact of successive exertional heat injuries on thermoregulatory and systemic inflammatory responses in mice.

The purpose of the study was to determine if repeated exertional heat injuries (EHIs) worsen the inflammatory response. We assessed the impact of a single EHI bout (EHI0) or two separate EHI episodes separated by 1 (EHI1), 3 (EHI3), and 7 (EHI7) days in male C57BL/6J mice (n = 236). To induce EHI, mice underwent a forced running protocol until loss of consciousness or core temperature reached ≥ 42.7°C. Blood and tissue samples were obtained 30 min, 3 h, 1 day, or 7 days after the EHI. We observed that mice undergoing repeated EHI (EHI1, EHI3, and EHI7) had longer running distances before collapse (∼528 m), tolerated higher core temperatures (∼0.18°C higher) before collapse, and had higher minimum core temperature (indicative of injury severity) during recovery relative to EHI0 group (∼2.18°C higher; all P < 0.05). Heat resilience was most pronounced when latency was shortest between EHI episodes (i.e., thermal load and running duration highest in EHI1), suggesting the response diminishes with longer recoveries between EHI events. Furthermore, mice experiencing a second EHI exhibited increased serum and liver HSP70, and lower corticosterone, FABP2, MIP-1ß, MIP-2, and IP-10 relative to mice experiencing a single EHI typically at 30 min to 3 h after EHI. Our findings indicate that an EHI event may initiate some adaptive processes that provide acute heat resilience to subsequent EHI conditions. NEW & NOTEWORTHY Mice undergoing repeated exertional heat injuries, within 1 wk of an initial heat injury, appear to have some protective adaptations. During the second exertional heat injury, mice were able to run longer and sustain higher body temperatures before collapse. Despite this, the mice undergoing a second exertional heat injury were more resilient to the heat as evidenced by attenuated minimum body temperature, higher HPS70 (serum and liver), lower corticosterone, and lower FABP2.

Influence of prior illness on exertional heat stroke presentation and outcome.

INTRODUCTION: Precipitating factors that contribute to the severity of exertional heat stroke (EHS) are unclear. The purpose of this study was to determine the effect of prior illness (PI) on EHS severity. METHODS: We performed a retrospective clinical record review of 179 documented cases of EHS at the Marine Corps Base in Quantico, Virginia. RESULTS: Approximately 30% of EHS cases had a medically documented PI. Anthropometrics (height, weight, body mass index) and commonly associated risk factors for EHS (age, number of days in training, wet bulb globe temperature, sleep patterns) did not differ between PI and no illness (NI) groups. PI patients presented with higher maximal rectal core temperatures (40.6 ± 1.0°C vs. 40.3 ± 1.2°C; P = 0.0419), and elevated pulse rates (118.1 ± 16.7 bpm vs. 110.5 ± 24.2 bpm; P = 0.0397). At the point of care, biomarker values were similar between PI and NI groups, with the exception of a trend toward elevated monocytes in those with PI (7.9 ± 2.9% vs 6.7± 2.7%; P = 0.0521). Rate and duration of cooling were similar between PI and NI patients. CONCLUSION: This study indicates that PI has a minimal effect on the patient presentation, severity and treatment outcome of EHS. The results of this study have important implications for military, civilian, and occupational populations who are at risk for EHS.

Pretreatment with indomethacin results in increased heat stroke severity during recovery in a rodent model of heat stroke.

It has been suggested that medications can increase heat stroke (HS) susceptibility/severity. We investigated whether the nonsteroidal anti-inflammatory drug (NSAID) indomethacin (INDO) increases HS severity in a rodent model. Core temperature (Tc) of male, C57BL/6J mice (n = 45) was monitored continuously, and mice were given a dose of INDO [low dose (LO) 1 mg/kg or high dose (HI) 5 mg/kg in flavored treat] or vehicle (flavored treat) before heating. HS animals were heated to 42.4°C and euthanized at three time points for histological, molecular, and metabolic analysis: onset of HS [maximal core temperature (Tc,Max)], 3 h of recovery [minimal core temperature or hypothermia depth (HYPO)], and 24 h of recovery (24 h). Nonheated (control) animals underwent identical treatment in the absence of heat. INDO (LO or HI) had no effect on physiological indicators of performance (e.g., time to Tc,Max, thermal area, or cooling time) during heating or recovery. HI INDO resulted in 45% mortality rate by 24 h (HI INDO + HS group). The gut showed dramatic increases in gross morphological hemorrhage in HI INDO + HS in both survivors and nonsurvivors. HI INDO + HS survivors had significantly lower red blood cell counts and hematocrit suggesting significant hemorrhage. In the liver, HS induced cell death at HYPO and increased inflammation at Tc,Max, HYPO, and 24 h; however, there was additional effect with INDO + HS group. Furthermore, the metabolic profile of the liver was disturbed by heat, but there was no additive effect of INDO + HS. This suggests that there is an increase in morbidity risk with INDO + HS, likely resulting from significant gut injury.NEW & NOTEWORTHY This paper suggests that in a translational mouse model, NSAIDs may be counterindicated in situations that put an individual at risk of heat injury. We show here that a small, single dose of the NSAID indomethacin before heat stroke has a dramatic and highly damaging effect on the gut, which ultimately leads to increased systemic morbidity.

Pathophysiology of blast-induced ocular trauma with apoptosis in the retina and optic nerve.

BACKGROUND: Blast-induced ocular trauma is a frequent cause of morbidity for survivors of improvised explosive devices. Blast overpressure (BOP) of 120 ± 7 KPa has been shown to cause damage to lungs, brain, and gut in a rat model; however, the effects of BOP on ocular tissues have not been characterized. To elucidate the pathophysiology of blast-induced ocular trauma, ocular tissues from rats subjected to blast were examined for evidence of apoptosis by the detection of activated caspase 3 and TUNEL assay in their ocular tissues. METHODS: A compressed air shock tube was used to deliver 120 ± 7 KPa of BOP for duration of 2 msec to the right side of the rats. Rats were then euthanized at specific time points after blast exposure (3 hours, 24 hours, 48 hours). Ocular tissues were processed for immunohistochemistry to detect activated caspase 3 and TUNEL assay. Tissues were evaluated for relative levels of positive signal as compared to nonblast exposed controls. RESULTS: Activated caspase 3 was detected in the optic nerve, ganglion layer, and inner nuclear layer post blast exposure. At 24 and 48 hours, the inner nuclear layer from the right side had more cells with activated caspase 3. In the optic nerve, the highest levels of activated caspase 3 were detected on the right side at 24 hours post blast. CONCLUSION: BOP of 120 ± 7 KPa induces optic neuropathy and retinal damage. In both the optic nerve and retina, caspase 3 was activated in the right and left sides following blast exposure. The results of this study reveal that blast exposure induces apoptosis in both the optic nerve and retinal tissues.

MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle.

PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE. METHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. RESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media. CONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.