Beneficial effects of activated macrophages on sulfur mustard-induced cutaneous burns, an in vivo experience.

OBJECTIVE: Macrophages are known to have key functions in almost every stage of wound healing and there is evidence for their beneficial effects in treating decubital ulcers and deep sternal wound infections in human. This study aimed to investigate the efficacy of a treatment with activated macrophages on ameliorating acute and long-term sulfur mustard (SM) induced skin injuries in the hairless guinea pig (HGP) model. METHODS: HGP were exposed to SM vapor and treated with either a single or multiple intra-dermal injections of human activated macrophages in suspension (hAMS) into the wound bed. Clinical and histological evaluations were conducted up to 4 weeks post-exposure. RESULTS: A single treatment with hAMS early after exposure (15 min and 6 h) resulted in a reduction in the number of damaged cells and vesications in the epidermis at 24 h. A substantial increase in cellular infiltration, mostly polymorphonuclears, was taking place in the hAMS-treated animals starting as early as 1 h after exposure. This flow of inflammatory cells continued, in the treated group, for at least 4 weeks, long after the injected macrophages were not detected. Repeated injections of hAMS (15 min, 48 h and 7 d post-exposure) decreased significantly the area of the wounds and improved the integrity of the barrier function as expressed by measuring trans-epidermal water loss up to 10 d. CONCLUSIONS: Our results indicate that the role of macrophages in wound healing is complex; their efficacy may depend on the timing of administration. Further investigation is required to determine whether they are required during the early phase of wound development and/or during the late phase of scar formation and remodeling.

Endothelial cell damage following sulfur mustard exposure in rabbits and its association with the delayed-onset ocular lesions.

OBJECTIVE: Ocular injuries following exposure to the toxic agent sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed corneal injuries, expressed clinically by neovascularization and epithelial defects. The present study aimed to investigate the effects of SM on corneal endothelium (CE) during the acute and delayed phase in relation to the development of the long-term pathology. METHODS: Rabbit eyes were exposed to SM vapor. A clinical follow-up including pachymetry for measurement of corneal thickness were conducted up to 3 months following exposure. In vivo analysis of corneal endothelium in the central and peripheral cornea was carried out, using a contact specular microscopy. Morphometric analysis of cell area and number of cells was performed, to include the acute and delayed phases. Eyes were taken for histology at different time points following exposure (1 h to 3 months). TUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling) was conducted for detection of apoptosis during the acute phase. RESULTS: SM induced acute corneal erosions and prolonged anterior segment inflammation. Corneal thickness increased within hours, declined after few days but remained higher compared to baseline value for months after the exposure, indicating a chronic edema. Apoptotic alterations were first observed at 6 h resulting in a significant decline in the number of endothelial cells at 24-48 h following exposure. Healing of the endothelium was relatively fast and at one week the Descemet's membrane was resurfaced, yet, the density and morphology of the cells was often abnormal. Moreover, histological evaluation revealed deformation and enlargement of many cells (polymegathism and pleomorphism), thickening and double layered Descemet's membrane. These changes were more pronounced in corneas displaying delayed pathology. DISCUSSION AND CONCLUSIONS: SM induced apoptotic cell death of endothelial cells that was accompanied by corneal edema. The impaired healing of the endothelium, including the decrease in endothelial cell density was associated with the delayed-onset injuries. Since human corneal endothelium is almost amitotic, endothelium toxicity should be taken into consideration when testing potential treatments against ocular injuries following SM exposure.

Characterization of acute and long-term pathologies of superficial and deep dermal sulfur mustard skin lesions in the hairless guinea pig model.

Sulfur mustard induces severe acute and prolonged damage to the skin and only partially effective treatments are available. We have previously validated the use of hairless guinea pigs as an experimental model for skin lesions. The present study aimed to characterize a model of a deep dermal lesion and to compare it with the previously described superficial lesion. Clinical evaluation of the lesions was conducted using reflectance colorimetry, trans-epidermal water loss and wound area measurements. Prostaglandin E(2) content, matrix metalloproteinase-2 and 9 activity, and histopathology were conducted up to 4 weeks post-exposure. Sulfur mustard skin injury, including erythema and edema, impairment of skin barrier and wounds developed in a dose-dependent manner. Prostaglandin E(2) content and matrix metalloproteinase-2 and 9 activities were elevated during the wound development and the healing process. Histological evaluation revealed severe damage to the epidermis and deep dermis and vesications. At 4 weeks postexposure, healing was not completed: significantly impaired stratum corneum, absence of hair follicles, and epidermal hyperplasia were observed. These results confirm the use of the superficial and deep dermal skin injuries in the hairless guinea pigs as suitable models that can be utilized for the investigation of the pathological processes of acute as well as long-term injuries. These models will be further used to develop treatments to improve the healing process and prevent skin damage and long-term effects.

Characterization of acute and long-term sulfur mustard-induced skin injuries in hairless guinea-pigs using non-invasive methods.

BACKGROUND/PURPOSE: Skin exposure to sulfur mustard (HD) results in erythema, edema and severe injury, which take long time to heal and might impose a heavy burden on the health system. Despite many years of research, there is no treatment that prevents the development of the cytotoxic effects of HD causing acute and prolonged damage to the skin. Therefore, it is of great importance to develop treatments that will ameliorate the extent of injury and improve as well as shorten the healing process. The aim of the present study was to establish a small animal model for a long-term HD-induced skin injury using the hairless guinea-pig (HGP) and to further test the efficacy of anti-inflammatories in ameliorating the pathology. METHODS: HGPs were exposed to HD vapor on four sites for various time durations (1, 5, 10, 15 and 30 min). Clinical evaluation was conducted using reflectance colorimetry, transepidermal water loss and wound-area measurements. Biochemical [prostaglandin (PGE) content and metalloproteinase-9 (MMP-9) activity] and histopathological evaluations were conducted up to 2 weeks post-exposure. RESULTS: Typical symptoms of HD skin injury developed including erythema and edema and the extent of injury was closely related to the exposure duration. Histological evaluation revealed severe edema, infiltration of inflammatory cells, damage to basal cells and vesication. By 2 weeks, healing was not completed, impaired basement membrane and epithelial hyperplasia were observed. PGE content and MMP-9 activity increased at 2 h post-exposure; however, while PGE returned to baseline levels within 24 h, MMP-9 remained elevated at least up to 48 h. Furthermore, a short-term, topical, anti-inflammatory post-exposure treatment was effective in reducing the extent of the acute injury. CONCLUSION: These results indicate that the effects of HD on HGP skin are similar to previously shown effects in the pig model and in humans and therefore support the use of the HGP as an animal model for long-term effects of HD on skin injury and for studying the efficacy of anti-inflammatory treatments.

Dermostyx (IB1) - High efficacy and safe topical skin protectant against percutaneous toxic agents.

Prevention of the penetration of toxic agents through the skin is crucial for both military troops and civilian populations. We have developed a novel topical skin protectant (TSP), coded as IB1 and commercially available as Dermostyx protective solution (Rekah Pharm, Israel). The formulation afforded significant protection against chemical warfare agents such as sulfur mustard (SM) and VX (2LD50), pesticides such as parathion and irritants such as acrolein. The efficacy of the protectant was evaluated in the pig model using clinical, histological and biochemical monitoring. A single topical application prior to exposure to the toxic agents reduced significantly the size and severity of skin lesions and ameliorated or prevented systemic clinical symptoms. The barrier properties of IB1 are immediate upon application and remain effective for at least 12 h. It is absorbed into the stratum corneum of the skin and remains there until rinsing with water, yet the ingredients are not absorbed into the body. The formulation is a hydrophilic water-based solution, composed of magnesium sulfate and glycerin that are widely used in cosmetic and medicine, and was shown to be safe in preclinical and in Phase I clinical studies. The suggested mode of action is based on the unique interaction of glycerin with the stratum corneum, changing its properties to hydrophilic and on the "salting out" effect of magnesium sulfate. The expected use of the TSP is by application on exposed skin areas and sensitive skin sites (e.g. armpits, groin, waist), when necessary. A quantity of 10 ml is sufficient for one application covering approximately 20% of the body surface area. The formulation was approved for human use by the Israel Ministry of Health and a CE mark certificate in Europe has been recently issued (Class I). Dermostyx has been adopted by the IDF and first responders as a skin protectant for special needs.

A topical skin protectant against chemical warfare agents.

BACKGROUND: Sulfur mustard and VX are potent chemical warfare agents that penetrate rapidly through the skin, causing severe prolonged injuries and sometimes death. OBJECTIVES: To develop a topically applied pretreatment that will act as a barrier and prevent the absorption of these agents through the skin, reducing morbidity and saving life. METHODS: Several formulations were developed and tested in preclinical animal studies in pigs. The protecting cream was applied as a single application (0.5-1 ml/100 cm2) prior to exposure (10 minutes to 12 hours) to sulfur mustard or VX. Assessment of sulfur mustard-induced skin damage was based on clinical and histologic evaluations. When tested against VX, clinical signs and blood cholinesterase activity were monitored. At the final stage of development, safety studies were conducted in animals and in human volunteers. RESULTS: The formulation that gave the best results, coded IB1 (under patent application), provided significant protection against a 1 hour exposure to sulfur mustard (droplets or vapor). All the pigs pretreated with IB1 cream survived a 1-4 hour challenge of 2xLD50 VX and did not exhibit any overt clinical signs. Protection was exhibited even when the cream was applied 12 hours (single application) prior to exposure. IB1 was found to be non-irritating in animals and humans. No adverse effects were found in a Phase I clinical study in young healthy volunteers when the cream was applied to around 20% of the skin surface (results presented elsewhere). CONCLUSIONS: IB1 cream has been shown to be a safe and effective topical skin protectant against the chemical warfare agents sulfur mustard and VX.

Anti-VEGF therapy (bevacizumab) for sulfur mustard-induced corneal neovascularization associated with delayed limbal stem cell deficiency in rabbits.

PURPOSE: To investigate the involvement of VEGF in corneal neovascularization (CNV) following sulfur mustard (SM) exposure and to test the therapeutic effects of bevacizumab (Avastin) in respect to dose, route of administration and timing. MATERIALS AND METHODS: Topical bevacizumab (6 or 25 mg/ml, ×2/day) was applied to rabbit eyes, before or after appearance of NV, following SM vapor exposure, and was compared with subconjunctival injection (25 mg/ml, ×2/week) and topical dexamethasone (1%, ×4/day). Treatments were given for 3 weeks. VEGF levels were monitored by immunohistochemistry and ELISA assay. Clinical evaluations included slit-lamp examination, impression cytology for diagnosis of Limbal Stem Cell Deficiency (LSCD), pachymetry, measurement of NV length and histology. RESULTS: Corneal NV was developed, as early as 2 weeks after exposure, in 50-70% of the eyes, associated with increased levels of VEGF. Topical bevacizumab treatment with both doses, starting at 4 weeks, reduced vascularization. Subconjunctival injection and topical dexamethasone were more potent. A combined treatment of dexamethasone and bevacizumab improved the anti-angiogenic efficacy, yet, there was no effect on LSCD. Topical bevacizumab treatment starting at 1 week, when VEGF was elevated but before appearance of NV, had no effect. CONCLUSIONS: VEGF was involved in corneal angiogenesis in SM-induced ocular injury. Bevacizumab was beneficial in reducing CNV by both, topical or subconjunctival injection, when given as a symptomatic therapy with or without dexamethasone, however with no effect on SC deficiency. Further studies on the pathological mechanism of SM-induced ocular surface disorder may direct towards improved therapy.

Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits.

PURPOSE: Ocular injuries following exposure to the chemical agent sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed Partial Limbal Stem Cell Deficiency (LSCD), expressed clinically by corneal neovascularization and epithelial defects. LSCD may derive from direct destruction of limbal stem cells or indirectly from altered limbal stromal niche. The aim of this study was to investigate the mechanism underlying LSCD in SM injuries, focusing on the effects of the chemical on limbal epithelium. METHODS: Rabbit eyes were exposed to SM vapor and were observed by slit lamp examinations and pachymetry. Eyes were taken for histological and molecular biology evaluations at different time points (4 h-4 weeks), to include acute and delayed injuries. Epithelial stem cells were identified by ABCG2, p63 and by in vivo BrdU labeling for slow cycling cells. RESULTS: Limbal stem cells were not damaged during the acute phase following SM exposure, in contrast to the severe injury of the central corneal epithelium. On the contrary, limbal epithelium became activated, responding to corneal insult with a wound healing process, as shown by histology and by transient elevation of the stem cells markers. Simultaneously, inflammation was taking place in the limbal stroma lasting for weeks. A gradual loss of stem cells was observed later-on (2-4 weeks), associated with typical symptoms of LSCD. CONCLUSIONS: LSCD associated with SM ocular toxicity was not derived from a direct cytotoxic effect on the epithelial stem cells, but apparently from pathological events at the limbal stroma, that produced an abnormal microenvironment for the stem cells, triggering their gradual death. The results, and in particular the absence of a primary damage to the epithelial stem cells, indicate the presence of a therapeutic window for intervention to avoid the development of the delayed LSCD.

Ocular injuries following sulfur mustard exposure--pathological mechanism and potential therapy.

Sulfur mustard (SM) is a potent vesicant, known for its ability to cause incapacitation and prolonged injuries to the eyes, skin and respiratory system. The toxic ocular events following sulfur mustard exposure are characterized by several stages: photophobia starting a few hours after exposure, an acute injury phase characterized by inflammation of the anterior segment and corneal erosions and a delayed phase appearing following a clinically silent period (years in human). The late injury appeared in part of the exposed eyes, expressed by epithelial defects and corneal neovascularization (NV), that lead to vision deficits and even blindness. During the last years we have characterized the temporal development of ocular lesions following SM vapor exposure in rabbits and have shown the existence of two sub-populations of corneas, those exhibiting delayed ocular lesions (clinically impaired) and those exhibiting only minor injuries if at all (clinically non-impaired). The aim of the present study was to investigate the pathological mechanism underlying the delayed injury by focusing on the unique characteristics of each sub-population and to test the efficacy of potential treatments. Clinically impaired corneas were characterized by chronic inflammation, increased matrix metalloproteinase (MMP) activity, poor innervation and limbal damage. Moreover, using impression cytology and histology, we identified the delayed lesions as typical for an ocular surface disorder under the category of limbal epithelial stem cell deficiency (LSCD). These results point to therapeutic directions, using anti-inflammatory drugs, MMPs inhibitors, neurotrophic factors and amniotic membrane transplantation. Topical anti-inflammatory drugs, either steroid (Dexamycin, DEX) or non-steroidal anti-infllammatory drug (NSAID, Voltaren Ophtha) were found to be beneficial in ameliorating the initial inflammatory response and in postponing the development of corneal NV, when given during the first week after exposure. When DEX was administered as a symptomatic treatment against NV, a significant regression in the angiogenic process was observed, however, the effect was temporal and blood vessels reappeared after therapy ceased. Chronic administration (8 weeks) of the MMP inhibitor Doxycycline was also effective in attenuation of the acute and delayed injury. Preliminary results, using amniotic membrane transplantation revealed some decrease of corneal edema with no effect on corneal NV. It is suggested that the chronic inflammation and prolonged impairment of corneal innervation are playing a role in the pathogenesis of the delayed LSCD following SM exposure by creating a pathological microenvironment to limbal epithelial stem cells, thus, leading to their slow death and to a second cascade of pathological events eventually resulting in severe long-term injuries. As of today, only topical anti-inflammatory drugs reached the criteria of an applicable efficient post-exposure ocular treatment for SM injuries. Further studies are required to investigate the effects of SM on epithelial stem cells and their involvement in the pathogenesis of the long-term injuries.

Subcellular alterations of protein kinase C isozymes in the rat brain after organophosphate poisoning.

The protein kinase C (PKC) signaling pathway has been associated with modulation of N-metyl-D-aspartate receptor activity, motor behavior, learning, and memory, all of which are severely impaired in organophosphate (OP) intoxication. Nevertheless, the role of PKC in OP intoxication is largely unknown. The present study attempted to characterize alterations in the immunoreactivity levels of PKC isozymes expressed in different brain areas in the rat following exposure to the nerve agent sarin (1x LD(50)). Furthermore, possible neuroprotective effect of selective PKC regulating peptide after such insult was evaluated. The results indicated that a significant reduction in the immunoreactivity level of the conventional betaII-PKC and the atypical zeta-PKC was observed in frontal cortex up to 24 h postsarin and in the striatum up to 5 days postsarin exposure. This reduction was in contrast to the increase in the immuno-reactivity level of both isozymes seen in the hippocampus or thalamus. Treatment with the anticonvulsant midazolam (0.5 mg/kg) 10 min postsarin exposure markedly reduced zeta-PKC immunoreactivity level and betaII-PKC in the membrane fractions in the hippocampus. betaII-PKC peptide (380 ng/kg), known to inhibit PKC translocation and activation, attenuated sarin-induced neuropathology. These observations suggest a role for both conventional and atypical PKC isozymes in OP-induced neuropathy in the rat and further support their involvement in cell death.

Amelioration of sulfur mustard skin injury following a topical treatment with a mixture of a steroid and a NSAID.

The ability to ameliorate sulfur mustard (HD)-induced oedema by treatment with anti-inflammatory drugs was reported previously after screening four steroids and four non-steroidal anti-inflammatory drugs (NSAIDs) using the mouse ear vesicant model. Following the screening study, one steroid and one NSAID (Adexone and Voltaren) were selected as the most effective, and a mixture of the two was chosen for the present more extensive research. The effect of the combined treatment on clinical, biochemical and histopathological parameters following HD insult was studied. Mice ears were exposed to 0.2 micro l of HD for 10 min to produce a moderate skin injury. Oedema development peaked ca. 48 h following exposure, as determined by weighing ear biopsies. Histological observations at that time exhibited damage to the epidermis and dermis. An increase in prostaglandin E (PGE) was measured in skin homogenates, starting 8 h following exposure and lasting at least up to 48 h post-exposure. A topical treatment using the above anti-inflammatory mixture significantly reduced inflammatory parameters when applied up to 4 h following exposure. These parameters included extent of oedema, levels of PGE, area of clinical damage and extent of cytotoxic injury (vesications and damaged epithelial cells). Thus, a combination of a steroid and NSAID was found to be effective in reducing the intensity of HD skin injury and possibly shortening the time to full recovery. The treatment, however, did not prevent completely the ensuing cytotoxic processes in the epithelial layer.