Immunopathology of terminal complement activation and complement C5 blockade creating a pro-survival and organ-protective phenotype in trauma.

BACKGROUND AND PURPOSE: Traumatic haemorrhage (TH) is the leading cause of potentially preventable deaths that occur during the prehospital phase of care. No effective pharmacological therapeutics are available for critical TH patients yet. Here, we identify terminal complement activation (TCA) as a therapeutic target in combat casualties and evaluate the efficacy of a TCA inhibitor (nomacopan) on organ damage and survival in vivo. EXPERIMENTAL APPROACH: Complement activation products and cytokines were analysed in plasma from 54 combat casualties. The correlations between activated complement pathway(s) and the clinical outcomes in trauma patients were assessed. Nomacopan was administered to rats subjected to lethal TH (blast injury and haemorrhagic shock). Effects of nomacopan on TH were determined using survival rate, organ damage, physiological parameters, and laboratory profiles. KEY RESULTS: Early TCA was associated with systemic inflammatory responses and clinical outcomes in this trauma cohort. Lethal TH in the untreated rats induced early TCA that correlated with the severity of tissue damage and mortality. The addition of nomacopan to a damage-control resuscitation (DCR) protocol significantly inhibited TCA, decreased local and systemic inflammatory responses, improved haemodynamics and metabolism, attenuated tissue and organ damage, and increased survival. CONCLUSION AND IMPLICATIONS: Previous findings of our and other groups revealed that early TCA represents a rational therapeutic target for trauma patients. Nomacopan as a pro-survival and organ-protective drug, could emerge as a promising adjunct to DCR that may significantly reduce the morbidity and mortality in severe TH patients while awaiting transport to critical care facilities.

Evaluation of the potential eye hazard at visible wavelengths of the supercontinuum generated by an ultrafast NIR laser in water.

Lasers with ultrashort pulse durations have become ubiquitous in various applications, including ocular surgery. Therefore, we need to consider the role of nonlinear optical effects, such as supercontinuum generation during propagation within the ocular media, when evaluating their potential hazard. We used a NIR femtosecond laser to generate a supercontinuum within an artificial eye. We recorded the visible spectra of the supercontinuum generated and calculated the energy contained within the visible band. Our results indicate that for wavelengths between 1350 nm and 1450 nm the energy contained within the visible band of the generated white light supercontinuum may surpass current safety exposure limits, and pose a risk of injury to the retina.

Development and Characterization of a Benchtop Corneal Puncture Injury Model.

During recent military operations, eye-related injuries have risen in frequency due to increased use of explosive weaponry which often result in corneal puncture injuries. These have one of the poorest visual outcomes for wounded soldiers, often resulting in blindness due to the large variations in injury shape, size, and severity. As a result, improved therapeutics are needed which can stabilize the injury site and promote wound healing. Unfortunately, current corneal puncture injury models are not capable of producing irregularly shaped, large, high-speed injuries as seen on the battlefield, making relevant therapeutic development challenging. Here, we present a benchtop corneal puncture injury model for use with enucleated eyes that utilizes a high-speed solenoid device suitable for creating military-relevant injuries. We first established system baselines and ocular performance metrics, standardizing the different aspects of the benchtop model to ensure consistent results and properly account for tissue variability. The benchtop model was evaluated with corneal puncture injury objects up to 4.2 mm in diameter which generated intraocular pressure levels exceeding 1500 mmHg. Overall, the created benchtop model provides an initial platform for better characterizing corneal puncture injuries as seen in a military relevant clinical setting and a realistic approach for assessing potential therapeutics.

Altered expression of aquaporin 1 and aquaporin 5 in the cornea after primary blast exposure.

Purpose: Our study aimed to determine whether the altered expression of biomarkers linked to corneal injuries, such as the edema-regulating proteins aquaporin-1 and aquaporin-5 (AQP1 and AQP5), occurred following primary blast exposure. Methods: Adult male Dutch Belted rabbits were anesthetized and exposed to blast waves with peak overpressures of 142.5-164.1 kPa (20.4-23.4 psi). These exposure groups experienced peak blast overpressure-specific impulses (impulse per unit surface area) of 199.6-228.5 kPa-ms. Unexposed rabbits were included as controls. The animals were euthanized at 48 h post-exposure. Corneas obtained from the euthanized blast-exposed and control rabbits were processed for quantitative PCR and western blot to quantify mRNA and the protein expression of AQP1 and AQP5. Immunohistochemical analysis was conducted to determine the cellular localization of AQP1 and AQP5. Results: Corneal thickness increased up to 18% with the peak blast overpressure-specific impulses of 199.6-228.5 kPa-ms at 48 h after blast exposure. mRNA levels of AQP1 and AQP5 increased in the whole cornea lysates of blast-exposed rabbits relative to those of the controls. Western blot analyses of whole cornea lysates revealed that the expression levels of AQP1 and AQP5 were approximately 2- and 1.5-fold higher, respectively, in blast-exposed rabbits compared to controls. The extent of AQP1 immunostaining (AQP1-IS) increased in the epithelial cell layer after blast exposure. The AQP5-IS pattern changed from a mixed membrane and cytoplasmic expression in the controls to predominantly cytoplasmic expression in the basally located cornea epithelial cells of blast-exposed rabbits. Conclusions: Primary blast exposure resulted in edema-related changes in the cornea manifested by the altered expression of the edema-regulating proteins AQP1 and AQP5 with blast overpressure-specific impulses. These findings support potential acute corneal injury mechanisms in which the altered regulation of water permeability is caused by primary blast exposure.

Blast Exposure Induces Ocular Functional Changes with Increasing Blast Over-pressures in a Rat Model.

Purpose: Blast-related brain and ocular injuries can lead to acute and chronic visual dysfunction. The chronic visual consequences of blast exposure and its progression remain unclear. The goal of this study is to analyze ocular functional response to four levels of blast exposure and identify a threshold of blast exposure leading to acute and chronic visual dysfunction. Methods: Anesthetized adult male Long-Evans rats received a single-blast exposure at a peak overpressure of 78, 117, 164 or 213 kPa, delivered by a compressed air-driven shock tube. Clinical eye examination, intraocular pressure (IOP), flash electroretinography (fERG) and spectral-domain optical coherence tomography (SD-OCT) images were assessed prior to, and at multiple time points post exposure. Results: No abnormal fERG were observed for the two lowest-level blast groups (78 kPa or 117 kPa). For the 164 kPa group, the a- and b-wave amplitudes of the fERG were decreased at 3 days postexposure (p = 0.009 for a-wave, p = 0.010 for b-wave), but recovered to baseline levels by 7 days post-exposure. The IOP was unchanged for the 117 kPa and 164 kPa groups. The 78 kPa group demonstrated a small transient increase during week one (p = 0.046). For the highest blast group (213 kPa), the IOP was significantly elevated immediately post-exposure (p = 0.0001), but recovered by 24 hr. A bimodal depression in the fERG a- and b-wave amplitudes was observed for this group: the amplitudes were depressed at day 3 post-exposure (p = 0.007 for a-wave, p = 0.012 for b-wave), and recovered by day 7 post-exposure. However, the fERG amplitudes were once again depressed at week 8 post-exposure, suggesting a chronic retinal dysfunction. All retinae appeared normal in SD-OCT images. Conclusions: Our study demonstrates that a single-blast exposure may result in acute and chronic fERG deficit, and traumatic IOP elevation. Noninvasive functional tests may hold promise for identifying individuals with a risk for developing chronic visual deficits, and indicating a time window for early clinical diagnosis, rehabilitation, and treatment.

Early Complement and Fibrinolytic Activation in a Rat Model of Blast-Induced Multi-Organ Damage.

OBJECTIVE: Blast injury is associated with multi-organ failure (MOF), causing significant morbidity and mortality in trauma patients. However, the pathogenesis of blast-induced MOF still remains obscure. In this study, we evaluate the pathophysiological changes related to blast-induced MOF in a clinically relevant rat model of blast injury. METHODS: A moderate blast overpressure was applied to induce injury in anesthetized rats. Pathological changes were evaluated by H&E staining. Complement activation, plasminogen, and myeloperoxidase levels were analyzed by complement hemolytic assay (CH50) and/or ELISA in blood samples. RESULTS: Analysis of lung, brain, and liver tissue at 24 hour after blast overpressure revealed severe injuries. The level of complement components C3 and C1q decreased in parallel with the reduction of CH50 level in injured animals at 1, 3, and 6 hours after blast. Consumption of plasminogen was also detected as early as 1 hour post-injury. Myeloperoxidase levels were elevated within 1 hour of blast injury. CONCLUSION: Our data reveal that blast injury triggers the complement and fibrinolytic systems, which likely contribute to blast-induced MOF. Conceivably, therapies that target these systems early may improve clinical outcomes in blast patients.

Repeat low-level blast exposure increases transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1) expression in the trigeminal ganglion.

Blast-associated sensory and cognitive trauma sustained by military service members is an area of extensively studied research. Recent studies in our laboratory have revealed that low-level blast exposure increased expression of transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1), proteins well characterized for their role in mediating pain transmission, in the cornea. Determining the functional consequences of these alterations in protein expression is critical to understanding blast-related sensory trauma. Thus, the purpose of this study was to examine TRPV1 and ET-1 expression in ocular associated sensory tissues following primary and tertiary blast. A rodent model of blast injury was used in which anesthetized animals, unrestrained or restrained, received a single or repeat blast (73.8 ± 5.5 kPa) from a compressed air shock tube once or daily for five consecutive days, respectively. Behavioral and functional analyses were conducted to assess blast effects on nocifensive behavior and TRPV1 activity. Immunohistochemistry and Western Blot were also performed with trigeminal ganglia (TG) to determine TRPV1, ET-1 and glial fibrillary associated protein (GFAP) expression following blast. Increased TRPV1, ET-1 and GFAP were detected in the TG of animals exposed to repeat blast. Increased nocifensive responses were also observed in animals exposed to repeat, tertiary blast as compared to single blast and control. Moreover, decreased TRPV1 desensitization was observed in TG neurons exposed to repeat blast. Repeat, tertiary blast resulted in increased TRPV1, ET-1 and GFAP expression in the TG, enhanced nociception and decreased TRPV1 desensitization.

Low-Level Blast Exposure Increases Transient Receptor Potential Vanilloid 1 (TRPV1) Expression in the Rat Cornea.

BACKGROUND: Blast-related ocular injuries sustained by military personnel have led to rigorous efforts to elucidate the effects of blast exposure on neurosensory function. Recent studies have provided some insight into cognitive and visual deficits sustained following blast exposure; however, limited data are available on the effects of blast on pain and inflammatory processes. Investigation of these secondary effects of blast exposure is necessary to fully comprehend the complex pathophysiology of blast-related injuries. The overall purpose of this study is to determine the effects of single and repeated blast exposure on pain and inflammatory mediators in ocular tissues. METHODS: A compressed air shock tube was used to deliver a single or repeated blast (68.0 ± 2.7 kPa) to anesthetized rats daily for 5 days. Immunohistochemistry was performed on ocular tissues to determine the expression of the transient receptor potential vanilloid 1 (TRPV1) channel, calcitonin gene-related peptide (CGRP), substance P (SP), and endothelin-1 (ET-1) following single and repeated blast exposure. Neutrophil infiltration and myeloperoxidase (MPO) expression were also assessed in blast tissues via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis, respectively. RESULTS: TRPV1 expression was increased in rat corneas exposed to both single and repeated blast. Increased secretion of CGRP, SP, and ET-1 was also detected in rat corneas as compared to control. Moreover, repeated blast exposure resulted in neutrophil infiltration in the cornea and stromal layer as compared to control animals. CONCLUSION: Single and repeated blast exposure resulted in increased expression of TRPV1, CGRP, SP, and ET-1 as well as neutrophil infiltration. Collectively, these findings provide novel insight into the activation of pain and inflammation signaling mediators following blast exposure.

Low-Level Primary Blast Causes Acute Ocular Trauma in Rabbits.

The objective of this study was to determine whether clinically significant ocular trauma can be induced by a survivable isolated primary blast using a live animal model. Both eyes of 18 Dutch Belted rabbits were exposed to various survivable low-level blast overpressures in a large-scale shock tube simulating a primary blast similar to an improvised explosive device. Eyes of the blast-exposed rabbits (as well as five control rabbits) were thoroughly examined before and after blast to detect changes. Clinically significant changes in corneal thickness arose immediately after blast and were sustained through 48 h, suggesting possible disruption of endothelial function. Retinal thickness (RT) increased with increasing specific impulse immediately after exposure. Intraocular pressure (IOP) was inversely correlated with the specific impulse of the blast wave. These findings clearly indicate that survivable primary blast causes ocular injuries with likely visual functional sequelae of clinical and military relevance.

Simulations of Porcine Eye Exposure to Primary Blast Insult.

PURPOSE: A computational model of the porcine eye was developed to simulate primary blast exposure. This model facilitates understanding of blast-induced injury mechanisms. METHODS: A computational model of the porcine eye was used to simulate the effects of primary blast loading for comparison with experimental findings from shock tube experiments. The eye model was exposed to overpressure-time histories measured during physical experiments. Deformations and mechanical stresses within various ocular tissues were then examined for correlation with pathological findings in the experiments. RESULTS: Stresses and strains experienced in the eye during a primary blast event increase as the severity of the blast exposure increases. Peak stresses in the model occurred in locations in which damage was most often observed in the physical experiments. CONCLUSIONS: Blast injuries to the anterior chamber may be due to inertial displacement of the lens and ciliary body while posterior damage may arise due to contrecoup interactions of the vitreous and retina. Correlation of modeling predictions with physical experiments lends confidence that the model accurately represents the conditions found in the physical experiments. TRANSLATIONAL RELEVANCE: This computational model offers insights into the mechanisms of ocular injuries arising due to primary blast and may be used to simulate the effects of new protective eyewear designs.

Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses.

The laser-induced retinal injury thresholds for repetitive-pulse exposures to 100-µs-duration pulses at a wavelength of 532 nm have been determined for exposures of up to 1000 pulses in an in vivo model. The ED50 was measured for pulse repetition frequencies of 50 and 1000 Hz. Exposures to collimated beams producing a minimal retinal beam spot and to divergent beams producing a 100-µm-diameter retinal beam spot were considered. The ED50 for a 100-µs exposure was measured to be 12.8 µJ total intraocular energy for a minimal retinal beam spot exposure and 18.1 µJ total intraocular energy for a 100-µm-diameter retinal beam spot. The threshold for exposures to N > 1 pulse was found to be the same for both pulse repetition frequencies. The variation of the ED50 with the number of pulses is described well by the probability summation model, in which each pulse is considered an independent event. This is consistent with a threshold-level damage mechanism of microcavitation for single-pulse 100-µs-duration exposures. The data support the maximum permissible exposure levels for repetitive-pulse exposure promulgated in the most recent laser safety guidelines.

Damage threshold from large retinal spot size repetitive-pulse laser exposures.

The retinal damage thresholds for large spot size, multiple-pulse exposures to a Q-switched, frequency doubled Nd:YAG laser (532 nm wavelength, 7 ns pulses) have been measured for 100 µm and 500 µm retinal irradiance diameters. The ED50, expressed as energy per pulse, varies only weakly with the number of pulses, n, for these extended spot sizes. The previously reported threshold for a multiple-pulse exposure for a 900 µm retinal spot size also shows the same weak dependence on the number of pulses. The multiple-pulse ED50 for an extended spot-size exposure does not follow the n dependence exhibited by small spot size exposures produced by a collimated beam. Curves derived by using probability-summation models provide a better fit to the data.

Anatomical manifestations of primary blast ocular trauma observed in a postmortem porcine model.

PURPOSE: We qualitatively describe the anatomic features of primary blast ocular injury observed using a postmortem porcine eye model. Porcine eyes were exposed to various levels of blast energy to determine the optimal conditions for future testing. METHODS: We studied 53 enucleated porcine eyes: 13 controls and 40 exposed to a range of primary blast energy levels. Eyes were preassessed with B-scan and ultrasound biomicroscopy (UBM) ultrasonography, photographed, mounted in gelatin within acrylic orbits, and monitored with high-speed videography during blast-tube impulse exposure. Postimpact photography, ultrasonography, and histopathology were performed, and ocular damage was assessed. RESULTS: Evidence for primary blast injury was obtained. While some of the same damage was observed in the control eyes, the incidence and severity of this damage in exposed eyes increased with impulse and peak pressure, suggesting that primary blast exacerbated these injuries. Common findings included angle recession, internal scleral delamination, cyclodialysis, peripheral chorioretinal detachments, and radial peripapillary retinal detachments. No full-thickness openings of the eyewall were observed in any of the eyes tested. Scleral damage demonstrated the strongest associative tendency for increasing likelihood of injury with increased overpressure. CONCLUSIONS: These data provide evidence that primary blast alone (in the absence of particle impact) can produce clinically relevant ocular damage in a postmortem model. The blast parameters derived from this study are being used currently in an in vivo model. We also propose a new Cumulative Injury Score indicating the clinical relevance of observed injuries.

Laser-induced retinal damage threshold measurements with wavefront correction.

An adaptive optics (AO) system was incorporated into a laser retinal exposure setup in order to correct for refractive error and higher-order aberrations of the nonhuman primate (NHP) eye during an in vivo retinal ED(50) measurement. Using this system, the ED(50) for a 100-ms, 532-nm small spot size exposure was measured to be 1.05 mJ total intraocular energy (TIE), a reduction of 22% from the value measured without aberration correction. The ED(50) for a 3.5-ns, 532-nm exposure was measured to be 0.51 microJ TIE, the lowest ED(50) reported for a ns-duration exposure. This is a reduction of 37% from the value measured without aberration correction and is a factor of only 2.6 higher than the maximum permissible exposure (MPE) for a 3.5-ns, visible wavelength small spot size exposure. The trend of in vitro measurements using retinal explants suggests that the in vivo ED(50) for small spot-size exposures could potentially be one order of magnitude smaller than the previously reported in vivo ED(50). Distortion of the incident laser beam by ocular aberrations cannot fully explain the discrepancy between the in vivo measurements with no aberration correction and the in vitro results.

Laser retinal thermal damage threshold: impact of small-scale ocular motion.

The impact of the small-scale ocular motion that occurs during steady gaze on the retinal thermal damage threshold for long-duration laser exposures is investigated. Exposure durations from 100 msec to 50 sec are considered. Experimentally recorded eye movement data are input into a numerical simulation to calculate the increase in temperature experienced by the retina during an exposure to a continuous wave laser. Calculations are for a small retinal beam spot. An Arrhenius damage integral is used to estimate the thermal damage threshold. The impact of the ocular movements is expressed as a relief factor chi, defined as the ratio of the theoretical damage threshold in the presence of ocular motion to the threshold calculated assuming no eye motion. The relief factor is found to be 1.05 for a 100-msec exposure, increasing to 1.3 for a 50-sec exposure. The relief factor is described well by the equation chi=1.12tau(0.037) for exposure durations tau in the range 100 msec to 50 sec.

Computer model to investigate the effect of eye movements on retinal heating during long-duration fixation on a laser source.

A computer simulation called RHME (Retinal Heating in Moving Eye) is developed to simulate the heating pattern that occurs in the retina during a long-duration exposure to a continuous wave laser beam. The simulation takes into account eye movements that occur during a deliberate fixation. Due to the rapid (millisecond) thermal time scale for heating and cooling, only the area of the retina directly exposed to the laser sustains an increased temperature. Once the laser spot is removed from a particular location of the retina (because of eye movements) that location quickly cools. Points of the retina will therefore have a complex thermal history during a long-duration exposure. Simulation results for a minimal retinal spot size indicate that subjects staring at a helium-neon laser (lambda=632.8 nm) beam producing the small-source maximum permissible exposure (MPE) level corneal irradiance of 1 mW cm(-2) (>10-s exposure) will experience a maximum although transient temperature increase in the retina of less than 2 degrees C during a 50-s fixation trial. The large increase in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) and ANSI Z136.1 safety limits for a long-duration small-source exposure to visible continuous wave lasers that was adopted in 2000 therefore appears appropriate.

Effect of source intensity on ability to fixate: implications for laser safety.

During long-term viewing of a continuous light source, head and eye movements affect the distribution of energy deposited in the retina. Previous studies of eye movements during a fixation task provided data used for revising the safety limits for long-term viewing of such sources. These studies have been continued to determine the effect of source brightness on the nature of fixational eye movements. Volunteers fixated for 50 s on a HeNe laser (lambda = 632.8 nm) masked by a small aperture to produce a target subtending approximately 0.03 mrad in the visual field. The source was attenuated to yield corneal irradiance values in the range 0.6 pW cm(-2) to 6 microW cm(-2). Eye movements were recorded using a Dual Purkinje Image Eyetracker. The data were characterized by fixation ellipses that represent areas of the retina in which the image of the spot was located 68% of the time of each trial. Significant variation across subjects in the tightness of fixation was observed. Over the eight orders of magnitude of source brightness used in this experiment (10(-13) to 10(-6) W cm(-2)), no subject showed more than roughly a factor of two variation in the area of the fixation ellipse. No statistically significant trend in tightness of fixation as a function of source brightness was observed. There was no loss of ability to fixate, nor any drive to aversion, at the higher source intensities.