Treatment of Sulfur Mustard Corneal Injury by Augmenting the DNA Damage Response (DDR): A Novel Approach.

Sulfur mustard (SM) is a highly reactive organic chemical has been used as a chemical warfare agent and terrorist threat since World War I. The cornea is highly sensitive to SM toxicity and exposure to low vapor doses can cause incapacitating acute injuries. Exposure to higher doses can elicit persistent secondary keratopathies that cause reduced quality of life and impaired or lost vision. Despite a century of research, there are no specific treatments for acute or persistent ocular SM injuries. SM cytotoxicity emerges, in part, through DNA alkylation and double-strand breaks (DSBs). Because DSBs can naturally be repaired by DNA damage response pathways with low efficiency, we hypothesized that enhancing the homologous recombination pathway could pose a novel approach to mitigate SM injury. Here, we demonstrate that a dilithium salt of adenosine diphosphoribose (INV-102) increases protein levels of p53 and Sirtuin 6, upregulates transcription of BRCA1/2, enhances γH2AX focus formation, and promotes assembly of repair complexes at DSBs. Based on in vitro evidence showing INV-102 enhancement of DNA damage response through both p53-dependent and p53-independent pathways, we next tested INV-102 in a rabbit preclinical model of corneal injury. In vivo studies demonstrate a marked reduction in the incidence and severity of secondary keratopathies in INV-102-treated eyes compared with vehicle-treated eyes when treatment was started 24 hours after SM vapor exposure. These results suggest DNA repair mechanisms are a viable therapeutic target for SM injury and suggest topical treatment with INV-102 is a promising approach for SM as well as other conditions associated with DSBs. SIGNIFICANCE STATEMENT: Sulfur mustard gas corneal injury currently has no therapeutic treatment. This study aims to show the therapeutic potential of activating the body's natural DNA damage response to activate tissue repair.

Dose-dependent emergence of acute and recurrent corneal lesions in sulfur mustard-exposed rabbit eyes.

Sulfur mustard (SM) is a lipid soluble alkylating agent that causes genotoxic injury. The eye is highly sensitive to SM toxicity and exposures exceeding 400 mg min/m3 can elicit irreversible corneal pathophysiologies. Development of medical countermeasures for ocular SM exposure has been hindered by a limited understanding of dose-dependent effects of SM on corneal injury. Here, clinical, histological and ultrastructural analyses were used to characterize the effects of SM dose on corneal injury progression. Corneas were evaluated for up to 20 wk following exposure to saturated SM vapor for 30-150 s, which corresponds to 300-1,500 mg min/m3. In acute studies, a ceiling effect on corneal edema developed at doses associated with full-thickness corneal lesions, implicating endothelial toxicity in corneal swelling. Recurrent edematous lesions (RELs) transiently emerged after 2 wk in a dose-dependent fashion, followed by the development of secondary corneal pathophysiologies such as neovascularization, stromal scarring and endothelial abnormalities. RELs appeared in 96 % of corneas exposed for ≥ 90 s, 52 % of corneas exposed for 60 s and 0 % of corneas exposed for 30 s. While REL latency was variable in corneas exposed for 60 s, REL emergence was synchronized at exposures ≥ 90 s. Corneas did not exhibit more than one REL, suggesting RELs are part of a programmed pathophysiological response to severe alkylating lesions. In post-mortem studies at 12 wk, corneal edema was positively correlated to severity of endothelial pathologies, consistent with previous findings that endothelial toxicity influences long-term outcomes. These results provide novel insight into long-term corneal pathophysiological responses to acute toxicity and identify exposure conditions suitable for therapeutic testing.

Corneal Endothelial Cell Toxicity Determines Long-Term Outcome After Ocular Exposure to Sulfur Mustard Vapor.

PURPOSE: Ocular exposure to sulfur mustard (SM) vapor causes acute loss of corneal endothelial cells (CECs). Persistent corneal endothelial pathologies are observed in eyes that do not recover from SM exposure, suggesting that endothelial toxicity contributes to mustard gas keratopathy (MGK). Here, we evaluated the contributions of endothelial loss to acute and chronic corneal injuries in SM-exposed eyes. METHODS: Rabbit eyes were exposed in vivo to equivalent doses of SM using 9-, 11-, or 14-mm vapor caps. The effects of exposure area on corneal injury progression were longitudinally evaluated over 12 weeks using clinical evaluations. The effects of exposure area on CEC morphology, endothelial and epithelial ultrastructure, and endothelial barrier function were determined from 1 day to 12 weeks. RESULTS: SM exposure caused loss of CECs and failure of endothelial barrier integrity at 1 day, independent of exposure cap size. By 3 weeks, eyes exposed with the 14-mm vapor cap exhibited increased corneal permeability, repopulation of the endothelium by cells with fibroblastic morphology, and abnormal deposition of extracellular matrix. Eyes exposed with 9- or 11-mm vapor caps exhibited transient symptoms of injury that fully resolved, with the rate of recovery correlated with cap size. CONCLUSIONS: The nonlinear correlation between endothelial lesion size and probability of developing MGK suggests that the CEC loss is a determinative factor for emergence of MGK. These studies illustrate the importance of endothelial repair in preventing MGK. Furthermore, they exclude chemical modification of basement membrane as a mechanistic cause of recurrent epithelial erosions in MGK eyes.

The physiology and toxicology of acute inhalation phosphine poisoning in conscious male rats.

Phosphine (PH3) is a toxidrome-spanning chemical that is widely used as an insecticide and rodenticide. Exposure to PH3 causes a host of target organ and systemic effects, including oxidative stress, cardiopulmonary toxicity, seizure-like activity and overall metabolic disturbance. A custom dynamic inhalation gas exposure system was designed for the whole-body exposure of conscious male Sprague-Dawley rats (250-350 g) to PH3. An integrated plethysmography system was used to collect respiratory parameters in real-time before, during and after PH3 exposure. At several time points post-exposure, rats were euthanized, and various organs were removed and analyzed to assess organ and systemic effects. The 24 h post-exposure LCt50, determined by probit analysis, was 23,270 ppm × min (32,345 mg × min/m3). PH3 exposure affects both pulmonary and cardiac function. Unlike typical pulmonary toxicants, PH3 induced net increases in respiration during exposure. Gross observations of the heart and lungs of exposed rats suggested pulmonary and cardiac tissue damage, but histopathological examination showed little to no observable pathologic changes in those organs. Gene expression studies indicated alterations in inflammatory processes, metabolic function and cell signaling, with particular focus in cardiac tissue. Transmission electron microscopy examination of cardiac tissue revealed ultrastructural damage to both tissue and mitochondria. Altogether, these data reveal that in untreated, un-anesthetized rats, PH3 inhalation induces acute cardiorespiratory toxicity and injury, leading to death and that it is characterized by a steep dose-response curve. Continued use of our interdisciplinary approach will permit more effective identification of therapeutic windows and development of rational medical countermeasures and countermeasure strategies.

Apoptotic cell death in rat lung following mustard gas inhalation.

To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in animals, we studied different caspases (caspase-8, -9, -3, and -6) in the lungs from a ventilated rat SM aerosol inhalation model. SM activated all four caspases in cells obtained from bronchoalveolar lavage fluid (BALF) as early as 6 h after exposure. Caspase-8, which is known to initiate the extrinsic Fas-mediated pathway of apoptosis, was increased fivefold between 6 and 24 h, decreasing to the unexposed-control level at 48 h. The initiator, caspase-9, in the intrinsic mitochondrial pathway of apoptosis as well as the executioner caspases, caspase-3 and -6, all peaked (P < 0.01) at 24 h; caspase-3 and -6 remained elevated, but caspase-9 decreased to unexposed-control level at 48 h. To study further the Fas pathway, we examined soluble as well as membrane-bound Fas ligand (sFas-L and mFas-L, respectively) and Fas receptor (Fas-R) in both BALF cells and BALF. At 24 h after SM exposure, sFas-L increased significantly in both BALF cells (P < 0.01) and BALF (P < 0.05). However, mFas-L increased only in BALF cells between 24 and 48 h (P < 0.1 and P < 0.001, respectively). Fas-R increased only in BALF cells by 6 h (P < 0.01) after SM exposure. Apoptosis in SM-inhaled rat lung specimens was also confirmed by both immunohistochemical staining using cleaved caspase-3 and -9 antibodies and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as early as 6 h in the proximal trachea and bronchi, but not before 48 h in distal airways. These findings suggest pathogenic mechanisms at the cellular and molecular levels and logical therapeutic target(s) for SM inhalation injury in animals.

Contributions of tissue-specific pathologies to corneal injuries following exposure to SM vapor.

Corneal injuries resulting from ocular exposure to sulfur mustard (SM) vapor are the most prevalent chemical warfare injury. Ocular exposures exhibit three distinct, dose-dependent clinical trajectories: complete injury resolution, immediate transition to a chronic injury, or apparent recovery followed by the subsequent development of persistent ocular manifestations. These latter two trajectories include a constellation of corneal symptoms that are collectively known as mustard gas keratopathy (MGK). The etiology of MGK is not understood. Here, we synthesize recent findings from in vivo rabbit SM vapor studies, suggesting that tissue-specific damage during the acute injury can decrement the regenerative capacities of corneal endothelium and limbal stem cells, thereby predisposing the cornea to the chronic or delayed forms of MGK. This hypothesis not only provides a mechanism to explain the acute and MGK injuries but also identifies novel therapeutic modalities to mitigate or eliminate the acute and long-term consequences of ocular exposure to SM vapor.

Interleukin-18 expression increases in response to neurovascular damage following soman-induced status epilepticus in rats.

BACKGROUND: Status epilepticus (SE) can cause neuronal cell death and impaired behavioral function. Acute exposure to potent acetylcholinesterase inhibitors such as soman (GD) can cause prolonged SE activity, micro-hemorrhage and cell death in the hippocampus, thalamus and piriform cortex. Neuroinflammation is a prominent feature of brain injury with upregulation of multiple pro-inflammatory cytokines including those of the IL-1 family. The highly pleiotropic pro-inflammatory cytokine interleukin-18 (IL-18) belongs to the IL-1 family of cytokines and can propagate neuroinflammation by promoting immune cell infiltration, leukocyte and lymphocyte activation, and angiogenesis and helps facilitate the transition from the innate to the adaptive immune response. The purpose of this study is to characterize the regional and temporal expression of IL -18 and related factors in the brain following SE in a rat GD seizure model followed by localization of IL-18 to specific cell types. METHODS: The protein levels of IL-18, vascular endothelial growth factor and interferon gamma was quantified in the lysates of injured brain regions up to 72 h following GD-induced SE onset using bead multiplex immunoassays. IL-18 was localized to various cell types using immunohistochemistry and transmission electron microscopy. In addition, macrophage appearance scoring and T-cell quantification was determined using immunohistochemistry. Micro-hemorrhages were identified using hematoxylin and eosin staining of brain sections. RESULTS: Significant increases in IL-18 occurred in the piriform cortex, hippocampus and thalamus following SE. IL-18 was primarily expressed by endothelial cells and astrocytes associated with the damaged neurovascular unit. The increase in IL-18 was not related to macrophage accumulation, neutrophil infiltration or T-cell appearance in the injured tissue. CONCLUSIONS: These data show that IL-18 is significantly upregulated following GD-induced SE and localized primarily to endothelial cells in damaged brain vasculature. IL-18 upregulation occurred following leukocyte/lymphocyte infiltration and in the absence of other IL-18-related cytokines, suggesting another function, potentially for angiogenesis related to GD-induced micro-hemorrhage formation. Further studies at more chronic time points may help to elucidate this function.

Structural, morphological, and functional correlates of corneal endothelial toxicity following corneal exposure to sulfur mustard vapor.

PURPOSE: Sulfur mustard (SM) is a highly reactive vesicant that causes severe ocular injuries. Following exposure to moderate or high doses, a subset of victims develops a chronic injury known as mustard gas keratopathy (MGK) involving a keratitis of unknown etiopathogenesis with secondary keratopathies such as persistent epithelial lesions, corneal neovascularization, and progressive corneal degeneration. This study was designed to determine whether SM exposure evokes acute endothelial toxicity and to determine whether endothelial pathologies were specifically observed in MGK corneas as opposed to healed corneas. METHODS: Corneas of New Zealand white rabbits were exposed to SM vapor, and the corneal endothelium was evaluated at 1 day and 8 weeks using scanning electron microscopy (SEM), transmission electron microscopy (TEM), in vivo confocal microscopy (IVM), and fluorescent microscopy. Barrier function was measured by uptake of a fluorescent dye injected into the anterior chamber. RESULTS: A centripetal endothelial injury at 1 day was observed by SEM, TEM, IVM, and fluorescent microscopy. In vivo confocal microscopy revealed additional cytotoxicity between 1 and 13 days. In contrast to healed corneas, which appeared similar to sham-exposed naive eyes at 8 weeks, MGK corneas exhibited significant evidence of continued pathological changes in the endothelium. CONCLUSIONS: Endothelial toxicity occurs at the right time and with the appropriate pathophysiology to contribute to MGK. Based on these findings, we propose a model that explains the relationships among SM dose, the biphasic progression, and the various clinical trajectories of corneal SM injury and that provides a mechanism for temporal variations in MGK onset. Finally, we discuss the implications for the management of SM casualties.

Novel application of stem cell-derived neurons to evaluate the time- and dose-dependent progression of excitotoxic injury.

Glutamate receptor (GluR)-mediated neurotoxicity is implicated in a variety of disorders ranging from ischemia to neural degeneration. Under conditions of elevated glutamate, the excessive activation of GluRs causes internalization of pathologic levels of Ca(2+), culminating in bioenergetic failure, organelle degradation, and cell death. Efforts to characterize cellular and molecular aspects of excitotoxicity and conduct therapeutic screening for pharmacologic inhibitors of excitogenic progression have been hindered by limitations associated with primary neuron culture. To address this, we evaluated glutamate-induced neurotoxicity in highly enriched glutamatergic neurons (ESNs) derived from murine embryonic stem cells. As of 18 days in vitro (DIV 18), ESNs were synaptically coupled, exhibited spontaneous network activity with neurotypic mEPSCs and expressed NMDARs and AMPARs with physiological current:voltage behaviors. Addition of 0.78-200 µM glutamate evoked reproducible time- and dose-dependent metabolic failure in 6 h, with a calculated EC50 value of 0.44 µM at 24 h. Using a combination of cell viability assays and electrophysiology, we determined that glutamate-induced toxicity was specifically mediated by NMDARs and could be inhibited by addition of NMDAR antagonists, increased extracellular Mg(2+) or substitution of Ba(2+) for Ca(2+). Glutamate treatment evoked neurite fragmentation and focal swelling by both immunocytochemistry and scanning electron microscopy. Presentation of morphological markers of cell death was dose-dependent, with 0.78-200 µM glutamate resulting in apoptosis and 3000 µM glutamate generating a mixture of necrosis and apoptosis. Addition of neuroprotective small molecules reduced glutamate-induced neurotoxicity in a dose-dependent fashion. These data indicate that ESNs replicate many of the excitogenic mechanisms observed in primary neuron culture, offering a moderate-throughput model of excitotoxicity that combines the verisimilitude of primary neurons with the flexibility and scalability of cultured cells. ESNs therefore offer a physiologically relevant platform that exhibits characteristic NMDAR responses, and appears suitable to evaluate molecular mechanisms of glutamate-induced excitotoxicity and screen for candidate therapeutics.

Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents.

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.

Architectural and biochemical expressions of mustard gas keratopathy: preclinical indicators and pathogenic mechanisms.

A subset of victims of ocular sulfur mustard (SM) exposure develops an irreversible, idiotypic keratitis with associated secondary pathologies, collectively referred to as mustard gas keratopathy (MGK). MGK involves a progressive corneal degeneration resulting in chronic ocular discomfort and impaired vision for which clinical interventions have typically had poor outcomes. Using a rabbit corneal vapor exposure model, we previously demonstrated a clinical progression with acute and chronic sequelae similar to that observed in human casualties. However, a better understanding of the temporal changes that occur during the biphasic SM injury is crucial to mechanistic understanding and therapeutic development. Here we evaluate the histopathologic, biochemical and ultrastructural expressions of pathogenesis of the chronic SM injury over eight weeks. We confirm that MGK onset exhibits a biphasic trajectory involving corneal surface regeneration over the first two weeks, followed by the rapid development and progressive degeneration of corneal structure. Preclinical markers of corneal dysfunction were identified, including destabilization of the basal corneal epithelium, basement membrane zone abnormalities and stromal deformation. Clinical sequelae of MGK appeared abruptly three weeks after exposure, and included profound anterior edema, recurring corneal erosions, basement membrane disorganization, basal cell necrosis and stromal degeneration. Unlike resolved corneas, MGK corneas exhibited frustrated corneal wound repair, with significantly elevated histopathology scores. Increased lacrimation, disruption of the basement membrane and accumulation of pro-inflammatory mediators in the aqueous humor provide several mechanisms for corneal degeneration. These data suggest that the chronic injury is fundamentally distinct from the acute lesion, involving injury mechanisms that operate on different time scales and in different corneal tissues. Corneal edema appears to be the principal pathology of MGK, in part resulting from persistent necrosis of the basal corneal epithelium and deterioration of the basement membrane. The findings also provide a potential explanation as to why administration of anti-inflammatories transiently delays, but does not prevent, the development of MGK sequelae.

Pathogenesis of acute and delayed corneal lesions after ocular exposure to sulfur mustard vapor.

PURPOSE: Sulfur mustard (SM) exposure results in dose-dependent morbidities caused by cytotoxicity and vesication. Although lesions resulting from ocular exposure often resolve clinically, an idiopathic delayed mustard gas keratopathy (MGK) can develop after a moderate or severe exposure. Sequelae include persistent keratitis, recurring epithelial lesions, corneal neovascularization, and corneal degeneration, which can lead to impaired vision or loss of sight. The purpose of this effort is to correlate structural changes with injury progression during the development of MGK. METHODS: New Zealand White rabbit corneas were exposed to SM using a vapor cup delivery system. The transition from acute to delayed injury was characterized by clinical, histological, and ultrastructural metrics over 8 weeks. RESULTS: Exposure dose was correlated to the likelihood of developing MGK but not to its severity. In a 56-animal cohort, a 2.5-minute exposure generated a corneal lesion, with 89% of corneas developing MGK within 5 weeks. A significant decrease in corneal edema at 2 weeks was predictive of the 11% of corneas that underwent asymptomatic recovery. Ultrastructural comparison of asymptomatic and MGK corneas at 8 weeks indicates that MGK is characterized by persistent edema and profound disorganization of the basement membrane zone. CONCLUSIONS: Ultrastructural changes associated with the delayed pathophysiology of corneal SM vapor exposure involve severe degeneration of the basement membrane zone and persistent edema. The mechanisms underlying MGK pathogenesis seem to alter injury progression as soon as 2 weeks after exposure. These data suggest that the vapor cup model system is suitable for therapeutic evaluation.

Role of acetylcholinesterase on the structure and function of cholinergic synapses: insights gained from studies on knockout mice.

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE(+/+)) or AChE KO (AChE(-/-)) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE(-/-) mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE(-/-) mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE(-/-) hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE(-/-) mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE(+/+) mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE(-/-) mice to function in the complete absence of AChE.

Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.

There are no pharmacological treatments to rescue botulinum neurotoxin (BoNT)-mediated paralysis of neuromuscular signaling. In part, this failure can be attributed to the lack of a cell culture model system that is neuron-based, allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis, yet still compatible with modern cellular and molecular approaches. We have developed a method to derive highly enriched, glutamatergic neurons from suspension-cultured murine embryonic stem (ES) cells. Hypothesizing that ES cell-derived neurons (ESNs) might comprise a novel platform to investigate the neurotoxicology of BoNTs, we evaluated the susceptibility of ESNs to BoNT/A and BoNT/E using molecular and functional assays. ESNs express neuron-specific proteins, develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions. They express the BoNT substrate SNARE proteins SNAP25, VAMP2 and syntaxin, and treatment with BoNT/A and BoNT/E holotoxin results in proteolysis of SNAP25 within 24 h with EC50s of 0.81 and 68.6 pM, respectively. Intoxication with BoNT/A results in the functional inhibition of potassium-induced, calcium-dependent glutamate release. ESNs remain viable and susceptible to intoxication for up to 90 days after plating, enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade. The evidence suggests that derived neurons are a novel, biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line, and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use.

Progression of ocular sulfur mustard injury: development of a model system.

Exposure of tissues to sulfur mustard (SM) results in the formation of protein and nucleotide adducts that disrupt cellular metabolism and cause cell death. Subsequent pathologies involve a significant proinflammatory response, disrupted healing, and long-term defects in tissue architecture. Following ocular exposure, acute corneal sequelae include epithelial erosions, necrosis, and corneal inflammation. Longer term, a progressive injury becomes distributed throughout the anterior chamber, which ultimately causes a profound remodeling of corneal tissues. In many cases, debilitating and vision-threatening injuries reoccur months to years after the initial exposure. Preliminary data in humans suffering from chronic epithelial lesions suggest that thymosin beta4 (Tbeta4) may be a viable candidate to mitigate acute or long-term ocular SM injury. To evaluate therapeutic candidates, we have developed a rabbit ocular exposure model system. In this paper, we report molecular, histological, ultrastructural, and clinical consequences of rabbit ocular SM injury, which can be used to assess Tbeta4 efficacy, including timepoints at which Tbeta4 will be assessed for therapeutic utility.

Microvesicating effects of sulfur mustard on an in vitro human skin model.

Bis-(beta-chloroethyl) sulfide (SM) is a potent skin vesicant previously used for chemical warfare. Progress in determination of the mechanistic basis of SM pathology, and development of prophylactic and/or therapeutic countermeasures to SM exposure has been hampered by lack of physiologically relevant models of human skin. The current work evaluated a newly developed tissue engineered full-thickness human skin model in a completely in vitro approach to investigation of SM-induced dermal pathology. The model was first characterized with regard to overall morphology, lipid composition, basement membrane (BM) composition and ultrastructural features that are important targets of SM pathologic activity. Well-developed BM ultrastructural features were observed at the dermal-epidermal junction (DEJ), thus demonstrating successful resolution of a primary deficiency of models previously evaluated for SM studies. Studies were then conducted to evaluate histopathological effects of SM on the model. Good replication of in vivo effects was observed, including apoptosis of basal keratinocytes (KC) and microblister formation at the DEJ. Tissue engineered skin models with well-developed basement membrane structures thus appear to be useful tools for in vitro mechanistic studies of SM vesicant activity and development of preventive/therapeutic approaches for SM pathology.

Gene expression profiling of rat hippocampus following exposure to the acetylcholinesterase inhibitor soman.

Soman (O-pinacolyl methylphosphonofluoridate) is a potent neurotoxicant. Acute exposure to soman causes acetylcholinesterase inhibition, resulting in excessive levels of acetylcholine. Excessive acetylcholine levels cause convulsions, seizures, and respiratory distress. The initial cholinergic crisis can be overcome by rapid anticholinergic therapeutic intervention, resulting in increased survival. However, conventional treatments do not protect the brain from seizure-related damage, and thus, neurodegeneration of soman-sensitive brain areas is a potential postexposure outcome. We performed gene expression profiling of the rat hippocampus following soman exposure to gain greater insight into the molecular pathogenesis of soman-induced neurodegeneration. Male Sprague-Dawley rats were pretreated with the oxime HI-6 (l-(((4-aminocarbonyl)pyridinio)methoxyl)methyl)-2-((hydroxyimino)methyl)-pyridinium dichloride; 125 mg/kg, ip) 30 min prior to challenge with soman (180 microg/kg, sc). One minute after soman challenge, animals were treated with atropine methyl nitrate (2.0 mg/kg, im). Hippocampi were harvested 1, 3, 6, 12, 24, 48, 72, 96, and 168 h after soman exposure and RNA extracted to generate microarray probes for gene expression profiling. Principal component analysis of the microarray data revealed a progressive alteration in gene expression profiles beginning 1 h postexposure and continuing through 24 h postexposure. At 48 h to 168 h postexposure, the gene expression profiles clustered nearer to controls but did not completely return to control profiles. On the basis of the principal component analysis, analysis of variance was used to identify the genes most significantly changed as a result of soman at each postexposure time point. To gain insight into the biological relevance of these gene expression changes, genes were rank ordered by p-value and categorized using gene ontology-based algorithms into biological functions, canonical pathways, and gene networks significantly affected by soman. Numerous signaling and inflammatory pathways were identified as perturbed by soman. These data provide important insights into the molecular pathways involved in soman-induced neuropathology and a basis for generating hypotheses about the mechanism of soman-induced neurodegeneration.

Medical management of cutaneous sulfur mustard injuries.

BACKGROUND: Sulfur mustard (2,2'-dichlorodiethyl sulfide; HD) is a potent vesicating chemical warfare agent that poses a continuing threat to both military and civilian populations. Significant cutaneous HD injuries can take several months to heal, necessitate lengthy hospitalizations, and result in long-term complications. There are currently no standardized or optimized methods of casualty management. New strategies are needed to provide for optimal and rapid wound healing. OBJECTIVE: The primary aim of this research was to develop improved clinical strategies (treatment guidelines) for optimal treatment of superficial dermal (second degree) cutaneous HD injuries, with the goal of returning damaged skin to optimal appearance and normal function in the shortest period of time. METHODS: Superficial dermal HD injuries were created on the ventral abdominal surface of weanling pigs. At 48h post-exposure, lesions were laser debrided and a treatment adjunct applied. Cultured epithelial allografts and 11 commercial off-the-shelf (COTS) products were examined for their efficacy in improving wound healing of these injuries. Clinical evaluations and a variety of non-invasive bioengineering methods were used at 7 and 14 days post-surgery to follow the progress of wound healing and evaluate various cosmetic and functional properties of the wounds. Measurements included reflectance colorimetry to measure erythema; evaporimetry to examine transepidermal water loss as a method of evaluating barrier function; torsional ballistometry to evaluate the mechanical properties of skin firmness and elasticity; and two-dimensional high frequency ultrasonography (HFU) to monitor skin thickness (e.g., edema, scar tissue). Histopathology and immunohistochemistry were performed 14 days following surgery to examine structural integrity and quality of healing. Logical Decisions((R)) for Windows was used to rank the 12 treatment adjuncts that were studied. RESULTS: The most efficacious treatment adjuncts included (1) Vacuum Assisted Closure, V.A.C., involving application of topical negative pressure, (2) Amino-Plex Spray (biO(2) Cosmeceuticals International, Inc., Beverly Hills, CA), a nutritive cosmeceutical product that is designed to increase oxygen in cells, stimulate ATP synthesis, improve glucose transportation, stimulate collagen formation, and promote angiogenesis, and (3) ReCell Autologous Cell Harvesting Device (Clinical Cell Culture Americas LLC, Coral Springs, Florida), an innovative medical device that was developed to allow rapid harvesting of autologous cells from a thin split-thickness biopsy followed by spray application of a population of skin cells onto wounds within 30 min of collecting the biopsy, without the need of culturing the keratinocytes in a clinical laboratory. CONCLUSIONS: Complete re-epithelialization of debrided HD injuries in 7 days is possible. In general, shallow laser debridement through the basement membrane zone (100 microm) appears to provide better results than deeper debridement (400 microm) with respect to early re-epithelialization, cosmetic appearance, functional restoration, and structural integrity. Of the 12 treatment adjuncts examined, the most promising included Vacuum Assisted Closure, Amino-Plex Spray, and ReCell Autologous Cell Harvesting Device.

Improved wound healing of cutaneous sulfur mustard injuries in a weanling pig model.

OBJECTIVE: The objective was to examine the efficacy of several treatment regimens in improving wound healing of cutaneous sulfur mustard (HD) injuries. METHODS: Wound healing studies were conducted in weanling pigs. Superficial dermal HD injuries were debrided at 48 hours postexposure using an erbium-doped yttrium aluminum garnet (Er:YAG) laser, followed by application of a treatment adjunct. A variety of noninvasive bioengineering methods were conducted during the postsurgical observation period to examine the various cosmetic and functional aspects of the skin. Histopathology was performed at the end of each study (14 or 21 days postsurgery). RESULTS: As noted clinically, reepithelialization was nearly complete by 7 days postsurgery for many of the sites treated with petrolatum and scarlet red dressings. By 21 days, the skin elasticity of the petrolatum-dressed sites was not significantly different from that of sham-exposed skin. Upon dressing removal on postsurgery day 4, the neoepidermis of allograft- and thin film-dressed sites was partially removed, with resultant petechial hemorrhaging. Mean pathology scores for hydrocolloid-dressed sites were significantly lower than those of untreated HD-exposed sites on postsurgery day 14. CONCLUSIONS: Care must be taken during bandage changes, and a nonadherent dressing that could be left in place for a longer period of time (eg, 7 days) would be beneficial. The use of cultured epithelial allograft material may have a potential role if grown on a completely nonadherent backing and left undisturbed for at least a week. Xeroform Petrolatum and Scarlet Red Ointment dressings are effective and inexpensive treatment adjuncts for HD injuries.

Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents.

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer's disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning.