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Ocular injuries following sulfur mustard (SM) exposure are characterized by an acute phase expressed by corneal erosions and inflammation of the anterior segment that after a clinically silent period may be followed by irreversible corneal injuries. The latter includes epithelial defects, chronic inflammation and neovascularization (NV), and were defined in rabbits and in humans as Limbal Stem Cell Deficiency (LSCD), that derived from a delayed loss of corneal epithelial stem cells (ESC), due to secondary processes most likely in the epithelial stem cell (SC) niche. The present study expands our research on SM-induced ocular injury to rodents (rats and mice) following whole body vapor exposure, aiming to define whether the delayed development of LSCD is a general characteristic of SM ocular toxicity. Freely moving rats and mice were exposed to SM vapor (155 μg/l, 10 min). Clinical examination was carried out in rats and included a slit-lamp bio-microscopy, up to 6 months. Eyes were taken for histology at different time points following exposure and evaluation included hematoxylin and eosin (HE) staining for general morphology, PAS for identification of goblet cells and p63 immunohistochemistry for progenitor epithelial cells. Whole body exposure to SM vapor in rats and mice resulted in acute ocular injury characterized by corneal erosions and ocular inflammation. Following a brief recovery period, 80-90% of the exposed eyes developed corneal NV associated with abnormal corneal epithelium, stromal inflammation and endothelial damage. The late injury was accompanied by migration of conjunctival goblet cells to the cornea and a loss of limbal epithelial progenitor cells, indicating LSCD. The long-term ocular injury shown hereby in rats and mice was consistent with the lesions described in rabbits and in human casualties and demonstrated the general phenomenon of limbal epithelial stem cells deficiency in SM ocular toxicity. The delayed manifestation of this pathology points towards a therapeutic window for the development of medical countermeasures in small animals following exposure in a real life scenario.

Purpose: To further investigate the inflammatory response as one of the leading factors in the development of delayed limbal stem cell deficiency, clinically manifested by corneal neovascularization , following sulfur mustard ocular exposure in rabbits and to evaluate potential therapy. Materials and methods: Right eyes of rabbits were exposed to sulfur mustard vapor. Clinical examination was performed and the growth of corneal blood vessels was evaluated. Inflammatory cells (neutrophils, macrophages, antigen-presenting cells and T cells) were identified immunohistochemically. Tacrolimus (Prograf, Teva, 5mg/ml) an immuno-modulator drug, was tested via sub-conjunctival injection (50µl), started either before or after the development of neovascularization (72h and 4weeks, respectively) given once a week for 3weeks.

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

Severe manifestations of COVID-19 are mostly restricted to people with comorbidities. Here we report that induced mild pulmonary morbidities render SARS-CoV-2-refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Notably, the deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel mice-based animal model for the study of comorbidity-dependent severe COVID-19.

The use of sulfur mustard (SM) in global terrorism is still a relevant threat to both civilian population and military personnel. Casualties exposed to SM may present mild, moderate or severe acute ocular lesions followed by a complete ocular resolution, chronic lesions or re-emerged ocular pathologies after a latent period. Current treatment for SM-induced ocular injury is based mainly on the clinical manifestation at the different stages of the injury and includes pharmaceutical and surgical interventions. These therapeutic measures are beneficial but not sufficient, and the ocular injury remains a continuous challenge for medical professionals. This review focuses on treatment experience carried out in humans and studied in animal models, for both SM-induced ocular acute injury and late pathology. In general, therapeutic measures are based on clinical features of the ocular injury or on the involvement of specific factors during the ocular injury that point out towards potential treatments. Anti-inflammatory treatments and limbal stem cell transplantation techniques were developed based on the clinical manifestation of the ocular injury. Optional therapies for impaired corneal innervation and endothelium are suggested for future research. Additionally, studies on potential treatments with anti-matrix metalloproteinase (MMP), anti-vascular endothelial growth factor (VEGF) and anti-IL-6 agents are discussed. Consequently, future studies may reveal the potential of additional pharmacological and biological treatments or advanced cellular and molecular biology methods to serve as novel therapeutic measures and techniques for this complicated ocular injury.

Sulfur mustard (HD) and VX are potent chemical warfare agents (CWAs) that penetrate rapidly through the skin causing severe acute and prolonged injuries that might result in death. Moreover, several toxic industrial chemicals (TICS) that are being routinely used in the industry and agriculture can be as deadly and damaging. Thus, following percutaneous skin exposure, rapid removal of the toxic agent by decontamination is one of the most crucial components in the individual defense. This can be achieved by passive decontamination or by neutralization of the chemical with reactive decontaminants. Fuller’s earth powder is a well-known adsorbent, known for years as an efficient decontaminant. The aim of this chapter is to evaluate the efficacy of decontamination with Fuller’s earth powder using the pig animal model and to compare it to other common decontaminants. The experimental design included exposure to 1-μl droplets of neat HD or VX (2LD50) on the pigs’ back skin for various exposure durations up to 1 hour for HD and 5 hours for VX. The efficacy of the various decontaminants (water, M291, M295, RSDL) was evaluated using clinical evaluation and measurement of lesion area or extent of erythema following exposure to HD and clinical evaluation and measurements of cholinesterase (ChE) inhibition following exposure to VX. Fuller’s earth and RSDL were most efficient and yielded similar results under the same experimental conditions. Similar high efficacy was observed when decontamination was performed up to 30 minutes after HD skin exposure and up to 2 hours following exposure to VX. Due to the advantages of Fuller’s earth, being most efficient, safe, easy to use, inexpensive, and with long shelf life, it is recommended as a universal decontaminant which is suitable for both military personnel and civilians.

Eye exposure to organophosphate (OP) chemical warfare irreversible acetylcholinesterase inhibitors, results in long-term miosis and impaired visual function. In contrast to the well-documented miotic and ciliary muscle spasm observed following chemical warfare, OP ocular exposure, little is known regarding the ocular surface histopathological insult. The aim of the present study was to determine the degree of the ocular surface insult following sarin or VX ocular exposure and to evaluate potential anti-cholinergic treatments in counteracting this insult. Rats that were whole body exposed to various sarin concentrations (0.049–43 μg/L; 5 min exposure), showed a dose-dependent miotic response and light reflex impairment. Following whole body sarin exposure, a dose dependent ocular surface histopathological insult was developed. A week following exposure to a low concentration of 0.05 μg/L, conjunctival pathology was observed, while corneal insult was noticed only following exposure to a concentration of 0.5 μg/L and above. Both tissues presented poorer outcomes when exposed to higher sarin concentrations. In contrast, eyes topically exposed to 1 μg sarin demonstrated no ocular insult a week following exposure. On the contrary, topical exposure to 1 μg VX resulted in a significant corneal insult. Anticholinergic treatments such as 0.1% atropine or 2% homatropine, given shortly following VX exposure, counteracted this insult. The results of this study show that not only do anti-cholinergic treatments counteract the miotic response, but also prevent the histopathological insult observed when given shortly following OP exposure.

Exposure to sulfur mustard (SM) may result in severe ocular injuries. While some of the eyes show a clinical resolution of the injury (defined as clinically non-impaired), part of the eyes develop irreversible late ocular pathologies (defined as clinically impaired) that may lead to corneal blindness. Understanding the pathological mechanisms underlying the development of the late pathology may lead to improved treatment options. Therefore, this study aimed to investigate the mRNA expression profiles of corneas from clinically impaired, clinically non-impaired and naive eyes. Rabbit eyes were exposed to SM vapor and a clinical follow-up was carried out up to 4 weeks using a slit lamp microscope. At this time point, corneal tissues from clinically impaired, clinically non-impaired and naive eyes were processed for RNA sequencing (RNA-seq) and differential expression analyses. The differential expression profiles were further subjected to pathway enrichment analysis using Ingenuity Pathway Analysis (IPA). Real-time PCR was used for RNA-seq validation. The late pathology developed in 54%-80% of the eyes following ocular exposure to SM, clinically manifested by inflammation, corneal opacity and neovascularization. RNA-seq results showed significant differences in mRNA levels of hundreds of genes between clinically impaired, clinically non-impaired and naive corneas. Pathway enrichment analysis showed common pathways that were activated in all of the exposed eyes, such as Th1 and Th2 activation pathway, in addition to pathways that were activated only in the clinically impaired eyes compared to the clinically non-impaired eyes, such as IL-6 and ERK5 signaling. Corneal mRNA expression profiles for the clinically impaired, clinically non-impaired and naive eyes generated a comprehensive database that revealed new factors and pathways, which for the first time were shown to be involved in SM-induced late pathology. Our data may contribute to the research on both the pathological mechanisms that are involved in the development of the late pathology and the protective pathways that are activated in the clinically non-impaired eyes and may point out towards novel therapeutic strategies for this severe ocular injury.

Skin decontamination following exposure to chemical agents is a most important component of the individual defense doctrine, removing the agent, ceasing its penetration and preventing secondary contamination of the first responders. The goal of the current study was to compare the efficacy of Reactive Skin Decontaminant Lotion (RSDL) and Fuller's Earth (FE) following exposure to sulfur mustard (SM) and VX, aiming to find the optimal procedure for mass casualty decontamination protocol. Decontamination efficacy was evaluated in pigs by measurement of lesion area and erythema (SM) and cholinesterase inhibition and clinical symptoms (VX). FE and RSDL were highly effective against both agents. Following SM exposure, the two decontaminants demonstrated a significant decrease in lesions' size together with the decrease in exposure duration. Likewise, skin decontamination following exposure to VX with either FE or RSDL resulted in reduction in clinical symptoms and prevention of death. Decontamination was worthwhile even if postponed, up to 30 min (SM) and 2 h (VX). In conclusion, both decontamination products were efficient in ameliorating the toxic effects even though in a different mechanism. Finally, for mass casualty scenario, FE is preferred as a universal decontaminant, considering its safety, ease of use and longer shelf life.

Purpose The sight threatening sulfur mustard (SM) induced ocular injury presents specific symptoms for each clinical stage. The acute injury develops in all of the exposed eyes and is characterized by erosions and severe inflammation. The irreversible late pathology develops only in part of the eyes, and is clinically expressed by chronic inflammation and corneal neovascularization (NV). The mechanisms underlying this injury are still in research and treatment is insufficient. Aiming to shed light on pathological mechanisms and improve the therapeutic measures, we studied the expression pattern of various cytokines and chemokines at different clinical stages of the ocular injury. Methods Rabbit right eye was exposed to SM vapor and a clinical follow-up was carried out up to 4 weeks. Corneal and limbal tissues were collected at 48 h, 1w and 4w post exposure and IL-1α, IL-1β, IL-6, TNFα, macrophage chemotactic protein (MCP)-1 and IL-8 levels were measured by commercial ELISA kits. Results SM exposed eyes presented an acute injury that was partially resolved within a week in all of the exposed eyes, and was followed by an irreversible late pathology in 50%–80% of the eyes, beginning at 2w. A significant elevation was seen in levels of the studied factors, however each factor presented a unique expression pattern. At the peak of the acute injury, at 48 h, significantly higher levels of corneal IL-1α, IL-8, and TNFα and limbal IL-1α and MCP-1 were found compared to naive eyes. At 1w, corneal IL-1β, IL-6, IL-8 and TNFα and limbal IL-8 and MCP-1 levels were significantly higher compared to naive eyes. During the late pathology, at 4w, elevated levels of corneal IL-1β, IL-6 and MCP-1 and limbal MCP-1 and IL-8 were found only in eyes presenting NV. Conclusions The levels of the studied factors changed throughout the dynamic course of the ocular injury. The prolonged increased levels of limbal MCP-1 and IL-8 may contribute to the continuous recruitment of inflammatory cells, characterizing the symptoms of the late pathology. The significantly elevated IL-1β and IL-6 at 1w, after the resolution of the acute injury but before the clinical manifestation of the late pathology suggests a therapeutic window for intervention with prevention therapy. Mapping the expression pattern of these cytokines and chemokines points out towards stage-specific therapeutic options.

Sarin poisoned rats display a hyper-cholinergic activity including hypersalivation, tremors, seizures and death. Here we studied the time and dose effects of midazolam treatment following nerve agent exposure. Rats were exposed to sarin (1.2 LD50, 108 μg/kg, im), and treated 1 min later with TMB4 and atropine (TA 7.5 and 5 mg/kg, im, respectively). Midazolam was injected either at 1 min (1 mg/kg, im), or 1 h later (1 or 5 mg/kg i.m.). Cortical seizures were monitored by electrocorticogram (ECoG). At 5 weeks, rats were assessed in a water maze task, and then their brains were extracted for biochemical analysis and histological evaluation. Results revealed a time and dose dependent effects of midazolam treatment. Rats treated with TA only displayed acute signs of sarin intoxication, 29% died within 24h and the ECoG showed seizures for several hours. Animals that received midazolam within 1 min survived with only minor clinical signs but with no biochemical, behavioral, or histological sequel. Animals that lived to receive midazolam at 1h (87%) survived and the effects of the delayed administration were dose dependent. Midazolam 5 mg/kg significantly counteracted the acute signs of intoxication and the impaired behavioral performance, attenuated some of the inflammatory response with no effect on morphological damage. Midazolam 1mg/kg showed only a slight tendency to modulate the cognitive function. In addition, the delayed administration of both midazolam doses significantly attenuated ECoG compared to TA treatment only. These results suggest that following prolonged seizure, high dose midazolam is beneficial in counteracting adverse effects of sarin poisoning.

Introduction Magnetic resonance (MR) imaging is a sensitive modality for demonstrating in vivo alterations in brain structure and function after acute organophosphate (OP) poisoning. The goals of this study were to explore early imaging findings in organophosphate-poisoned animals, to assess the efficacy of centrally acting antidotes and to find whether early MR findings can predict post-poisoning cognitive dysfunction. Methods Sprague–Dawley rats were poisoned with the agricultural OP paraoxon and were treated with immediate atropine and obidoxime (ATOX) to reduce acute mortality caused by peripheral inhibition of acetylcholinesterase. Animals were randomly divided into three groups based on the protocol of centrally acting antidotal treatment: group 1 – no central antidotal treatment (n = 10); group 2 – treated with midazolam (MID) at 30 min after poisoning (n = 9), group 3 – treated with a combination of MID and scopolamine (SCOP) at 30 min after poisoning (n = 9) and controls (n = 6). Each animal had a brain MR examination 3 and 24 h after poisoning. Each MR examination included the acquisition of a T2 map and a single-voxel 1H MR spectroscopy (localized on the thalami, to measure total creatine [Cr], N-acetyl-aspartate [NAA] and cholines [Cho] levels). Eleven days after poisoning each animal underwent a Morris water maze to assess hippocampal learning. Eighteen days after poisoning, animals were euthanized, and their brains were dissected, fixed and processed for histology. Results All paraoxon poisoned animals developed generalized convulsions, starting within a few minutes following paraoxon injection. Brain edema was maximal on MR imaging 3 h after poisoning. Both MID and MID + SCOP prevented most of the cortical edema, with equivalent efficacy. Brain metabolic dysfunction, manifested as decreased NAA/Cr, appeared in all poisoned animals as early as 3 h after exposure (1.1 ± 0.07 and 1.42 ± 0.05 in ATOX and control groups, respectively) and remained lower compared to non-poisoned animals even 24 h after poisoning. MID and MID + SCOP prevented much of the 3 h NAA/Cr decrease (1.22 ± 0.05 and 1.32 ± 0.1, respectively). Significant correlations were found between imaging findings (brain edema and spectroscopic changes) and clinical outcomes (poor learning, weight loss and pathological score) with correlation coefficients of 0.4–0.75 (p Conclusions MR imaging is a sensitive modality to explore organophosphate-induced brain damage. Delayed treatment with midazolam with or without scopolamine provides only transient neuroprotection with some advantage in adding scopolamine. Early imaging findings were found to correlate with clinical consequences of organophosphate poisoning and could be potentially used in the future to predict long-term prognosis of poisoned casualties.

Purpose To evaluate the efficacy of ziv-aflibercept as a treatment for established corneal neovascularization (NV) and to compare its efficacy to that of bevacizumab following ocular chemical insult of sulfur mustard (SM) in the rabbit model. Methods Chemical SM burn was induced in the right eye of NZW rabbits by vapor exposure. Ziv-aflibercept (2 mg) was applied once to neovascularized eyes by subconjunctival injection while subconjunctival bevacizumab (5 mg) was administered twice a week, for 3 weeks. Non-treated exposed eyes served as a control. A clinical follow-up employed by slit-lamp microscope, was performed up to 12 weeks following exposure and digital photographs of the cornea were taken for measurement of blood vessels length using the image analysis software. Eyes were taken for histological evaluation 2, 4 and 8 weeks following treatment for general morphology and for visualization of NV, using H&E and Masson Trichrome stainings, while conjunctival goblet cell density was determined by PAS staining. Results Corneal NV developed, starting as early as two weeks after exposure. A single subconjunctival treatment of ziv-aflibercept at 4 weeks post exposure, significantly reduced the extent of existing NV already one week following injection, an effect which lasted for at least 8 weeks following treatment, while NV in the non-treated exposed eyes continued to advance. The extensive reduction in corneal NV in the ziv-aflibercept treated group was confirmed by histological evaluation. Bevacizumab multiple treatment showed a benefit in NV reduction, but to a lesser extent compared to the ziv-aflibercept treatment. Finally, ziv-aflibercept increased the density of conjunctival goblet cells as compared to the exposed non-treated group. Conclusions Subconjunctival ziv-aflibercept single treatment presented a highly efficient long-term therapeutic benefit in reducing existing corneal NV, following ocular sulfur mustard exposure. These findings show the robust anti-angiogenic efficacy of ziv-aflibercept and demonstrate the advantage of this treatment over the other anti-angiogenic therapies in ameliorating corneal NV and protecting the ocular surface.

Sulfur mustard (SM) is an incapacitating chemical warfare agent used in numerous conflicts around the world and it is still a major threat for both, army troops and civilians. To evaluate its multiple targets effects in experimental setup, a model of whole body exposure (WBE) to SM vapor was established in rats and its simultaneous effects on lungs and eyes as well as on general wellbeing were examined. Rats were exposed to SM vapor. Evaluation (up to 10 weeks post-exposure) included body weight, general observation, blood counts and histological analysis. Results showed that following a latency-period of several hours, rats typical symptoms developed over a period of more than one week. The initial symptoms, characterized by swollen and erythematic nose, deteriorated into extensive rhinorrhea, eye closure, excessive lacrimation as well as rhonchi, wheezing and breathing difficulties. Alopecia and behavioral abnormality were also recorded. A weight loss of up to 40% was measured within one week with spontaneous recovery to baseline level within three weeks after exposure. Blood counts revealed leukopenia during the first three days post-exposure. Histological evaluation revealed a long lasting damage to the trachea, lungs and eyes. Thus, WBE to SM, was found to closely mimic the deleterious effects of SM on the sensitive tissues previously described in human victims during WWI and the Iran-Iraq war. The use of this animal model will enable comprehensive characterization of changes in biological processes that may lead to the development of therapeutic measures to ameliorate SM induced multi-system injuries.

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.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.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.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.

We aimed to evaluate the performance of medical personnel in using the IB1 topical protective lotion on their hands and wrists together with standard disposable medical gloves, compared to standard-issued medical chemical protective gloves. This randomized cross-over study included 144 medical personnel. Primary endpoints were time-to-completion of autoinjection; success rate, number of attempts, and time-to-achieve successful endotracheal intubation; time-to-achieve satisfactory tube fixation; time-to-draw and inject the content of an ampoule; and the total time-to-perform all medical procedures. Secondary endpoints included the subjective assessment of convenience to perform these four procedures with each protective measure. Mean time was significantly shorter using IB1 compared to chemical protective gloves for tube fixation, ampoule drawing, and the total time-to-perform all procedures (58.6±22.7 seconds vs. 71.7±29.7; 31.5±21.8 vs. 38.2±19.4; 137.4±56.1 vs. 162.5±63.6, respectively; P.001 for all). For all medical procedures, the use of IB1 was reported as significantly more convenient than the use of chemical protective gloves (P.001 for all comparisons). IB1 with standard medical gloves significantly shorten the time-to-perform medical procedures requiring fine motor dexterities and is subjectively more convenient than chemical protective gloves. IB1 should be considered as an appropriate alternative for medical teams in a chemical event.

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.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.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.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.

Guinea pigs (GP) were exposed (head only) in individual plethysmographs to various concentrations of sulfur mustard vapor, determined online, using FTIR attached to flow chamber. The LCt(50) and the inhaled LD(50) were calculated at different time points post exposure. Surviving animals were monitored for clinical symptoms, respiratory parameters and body weight changes for up to 30 days. Clinical symptoms were noted at 3 h post exposure, characterized by erythematic and swelling nose with extensive mucous secretion (with or without bleeding). At 6 h post exposure most of the guinea pigs had breathing difficulties, rhonchi and dyspnea and few deaths were noted. These symptoms peaked at 48 h and were noted up to 8 days, associated with few additional deaths. Thereafter, a spontaneous healing was noted, characterized by recovery of respiratory parameters and normal weight gain with almost complete apparent healing within 2 weeks. Histopathological evaluation of lungs and trachea in the surviving GPs at 4 weeks post exposure revealed a dose-dependent residual injury in both lung and trachea expressed by abnormal recovery of the tracheal epithelium concomitant with a dose-dependent increase in cellular volume in the lungs. These abnormal epithelial regeneration and lung remodeling were accompanied with significant changes in protein, LDH, differential cell count and glutathione levels in the bronchoalveolar lavage (BAL). It is suggested that the abnormal epithelial growth and cellular infiltration into the lung as well as the continuous lung inflammation could cause recurrent lung injury similar to that reported for HD exposed human casualties.

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.

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.

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.

Sulfur mustard (2,2‐dichlorodiethyl sulfide; HD), is a potent alkylating agent which in liquid or vapor form is capable of causing severe injuries to skin and respiratory tract, and was shown to cause short‐ and long‐term ocular injuries. N‐Acetylcysteine (NAC) may act as a mucolytic agent, changing the “wetting” and scavenging properties of the cornea and thus the adhesion of HD. Moreover, NAC is a scavenger of HD, an antioxidant and a glutathione precursor, which was shown to reduce HD toxicity in various systems. The ocular distribution of 14C, after topical application of liquid 14C‐sulfur mustard (14C‐HD) to the rabbit cornea, and the role of NAC in reducing HD retention and toxicity are presented in this study. Groups of rabbits were exposed to 0.4 µL of liquid 14C‐HD, placed at the center of the cornea, with or without NAC treatment. Fifty µL NAC (10% aqueous solution) was topically applied, 10 minutes before and 10 minutes after HD exposure. Three time points were evaluated: 1, 6, and 24 hr after ...

In spite of several decades of research, no effective treatment to skin injuries following exposure to sulfur mustard (HD) has yet been found. In the present study, the mouse ear vesicant model was applied to awake mice in order to evaluate the efficiency of potential anti-inflammatory treatments in preventing HD-induced skin damages. Clinical follow-up and histological evaluation were used to characterize the injuries to the skin and to evaluate the efficiency of the drugs that were applied. Thus, the extent of mouse ear oedema and the histopathological changes following a single application of 0.2 or 1 L of neat HD for 10 min (representing moderate and severe lesions, respectively), were monitored. Typical HD skin lesions were observed including epithelial and dermal damage. The development of the injury in mouse ears was found to be very similar to that reported in human skin. Screening of post-exposure topical steroids and non-steroidal anti inflammatory drugs (NSAIDs) proved that HD-induced inflammation could be diminished significantly as long as the treatment was applied during the early stages following exposure. A combined application of these drugs approved to be particularly effective in reducing inflammation.

Stroke in humans is usually associated with advanced age. Nevertheless, almost all animal models of ischemic stroke are based on young animals. The present study was designed to assess the effect of age on the development of ischemic injury in a model of focal brain ischemia in rats. Two age groups of Wistar rats were used: young adult (3 months) and old (24–26 months). Under halothane anesthesia, polyethylene microspheres (50 μm in diameter) were injected into the left common carotid artery following a temporary occlusion of the external carotid artery. Sham-operated rats underwent the same procedure but were injected with an identical volume (100 μl) of saline only. Rats of both experimental groups displayed neurological impairment after surgery. However, contrary to expectation, the young rats were more affected than the old rats. Young rats displayed an abrupt 30% decrement in neurological functions in the first week and then showed a partial functional recovery into a 12% decrement from the second week on. Old rats developed the neurological impairment gradually over a 2-week period (6.3% in the first week and 11% in the second week and thereafter). One month later, rats were tested in a water maze task. Again, performance was more impaired in the young ischemic rats than in the old rats. Histological evaluation revealed more extensive neurological damage in young ischemic as compared to old rats. Thus, although increased age has a critical effect on the evolution of the neurological impairment following focal brain ischemia and stroke, its effects in the rat model were more pronounced in the young animals.

Acrolein is a potent irritant and a vesicant that was used by the French during WWI as the warfare agent named: "papite". Nowadays, it is produced in large amounts all over the world in the industry for the production of acrylic acid and for agriculture use as herbicide. The aim of this study was to characterize the effects of acute eye exposure to acrolein vapor and to evaluate the efficacy of a topical post-exposure combination treatment with a local anesthetic and a steroid.Rabbit eyes were exposed to three doses of acrolein vapor (low, intermediate and high) and treated topically with either 0.4% benoxinate hydrochloride (localin, for 2 h) or dexamethasone (dexamycin, for 6 days) or a combination of both drugs. Clinical follow-up using slit lamp examinations and histological evaluation was performed 4 weeks post-exposure.Acrolein vapor caused immediate eye closure with excess tearing, corneal erosions and severe inflammation of the anterior chamber. This was followed by corneal neovascularization (NV) starting as early as 1 week post-exposure. The damage to the eyes was long lasting, and at 4 weeks following exposure, significant pathological changes were observed. Immediate post-exposure application of the local anesthetic, localin, prevented the eye closure, and the dexamycin treatment reduced significantly the initial inflammation as well as the extent and incidence of corneal NV.Short-term eye exposure to the irritant acrolein may result in immediate eye closure and long lasting pathologies that could lead to blindness. An anti-inflammatory treatment combined with short-term application of a local anesthetic prevents incapacitation and might minimize significantly the extent of eye injuries.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. Recent findings suggest that retina of apoE4 mice have synaptic and functional impairments. We presently investigated the effects of apoE4 on retinal and choroidal vasculature and the possible role of VEGF in these effects. There were no histological differences between the retinal and choroidal vasculatures of naive apoE3 and apoE4 mice. In contrast, laserdriven choroidal injury induced higher levels of choroidal neovascularization (CNV) in apoE4 than in apoE3 mice. These effects were associated with an inflammatory response and with activation of the Muller cells and asrocytic markers gluthatione synthetase and GFAP, all of which were more pronounced in the apoE4 mice. CNV also induced a transient increase in the levels of the synaptic markers synaptophysin and PSD95 which were however similar in the apoE4 and apoE3 naive mice. Retinal and choroidal VEGF and apoE levels were lower in naive apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury. Retinal and choroidal VEGF and apoE levels were lower in naive apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury.

Sulfur mustard (SM) induces acute ocular lesions, including erosions and inflammation that may be followed by delayed injuries expressed by epithelial defects and neovascularization (NV). Based on the matrix metalloproteinases (MMPs) activity, we evaluated the clinical and biochemical effects of topical treatment with doxycycline, an MMP inhibitor, targeted to the various injury stages.Rabbit eyes were exposed to SM vapor. A clinical follow-up was carried out up to 2 months. Tear fluid and cornea samples were collected at different time points for measurements of MMPs activity by zymography. Efficacy of a post-exposure topical doxycycline (2 mg/ml in phosphate buffer saline, ×4/d), targeted to the different phases of the clinical injury, was evaluated.Elevated MMP-9 and MMP-2 activities were found in all corneas during the acute injury and in vascularized corneas during the delayed pathology. In the tear fluid, high MMP-9 activity and negligible MMP-2 activity were found in all the exposed eyes until after the appearance of the delayed pathology symptoms. Prolonged doxycycline treatment reduced MMP-9 activity in the tear fluid. During the acute phase, doxycycline treatment reduced corneal MMP-9 activity and the severity of the injury. Targeting the delayed pathology, doxycycline was clinically efficient only when treatment began before NV appearance.This in vivo study showed the involvement of MMP-9 and MMP-2 during different phases of the SM-induced ocular injury, and the potential of doxycycline treatment as a post exposure measure for reducing the acute injury and as a preventive therapy for ameliorating the delayed pathology. The tear fluid provided a non-invasive method for continuous follow-up of MMPs activity and revealed additional beneficial aspects of injury and the treatment.

Various animal models, involving different brain insults, lead to memory deficits, which can be measured using behavioral tests. In numerous studies, using five different experimental models in rats, we have found that cognitive dysfunction is invariably accompanied by hippocampal CA1 and CA3 pyramidal cells degeneration. However, of these two, the most affected area changes from one model to the other. The present manuscript describes and compares the morphological alterations within the hippocampus in the following experimental models: normal aging, hypoxia, prolonged corticosterone administration, brain ischemia and cholinesterase (ChE) inhibition. In all the above, many hippocampal neurons were severely damaged, however, CA3 pyramidal cells were mostly affected in normal aging and following hypobaric hypoxia, whereas CA1 cells were especially affected following corticosterone administration, global ischemia and ChE inhibition. Several mechanisms, which might be involved in the diverse courses of the lesions are being considered: cerebral oxygen and glucose, glutamate neurotoxicity and calcium involvement. It is anticipated that elucidation of the specific role of CA1 and CA3 hippocampal sub-fields in the various experimental models might help in understanding processes such as age-related neuronal degeneration and assist in their prevention.

Sustained high levels of corticosterone (CORT), one of the major stress-induced hormones in the rat, were suggested as generating 'accelerated brain aging' and were shown to induce both specific brain changes in the hippocampus and learning impairments in young and middle-aged Fischer-344 rats. Evidence that altered calcium (Ca) homeostasis may play a major role in brain aging has accumulated over the last decade. Recently, new data established a connection between glucocorticoids and voltage-activated Ca influx in aged hippocampal neurons. In the present study, an attempt was made to block the CORT-induced 'accelerated aging' by the simultaneous administration of the L-type Ca channel blocker nimodipine. CORT or placebo sustained-release (SR) pellets were implanted subcutaneously in 3 months old Fischer male rats. Each group was further sub-divided between nimodipine and placebo SR treatments. Characteristic CORT-induced morphological changes were observed in pyramidal hippocampal cells, such as at the CA1 and CA4 sub-regions (22.2% +/- 7.7 and 28.6% +/- 8.4 of pyknotic cells without clear nuclei, respectively). Concomitant treatment with nimodipine conferred full protection against CORT-induced morphological changes (e.g. 3.2% +/- 0.8 and 2.1% +/- 1.9 of pyknotic cells in CA1 and CA4, n = 7 rats in each group; P0.04). The neuroprotective efficacy of nimodipine supports the theory of Ca involvement in CORT related 'accelerated brain aging'.

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.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.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.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.

Purpose Limbal epithelial sheets are used to promote corneal surface reconstruction after the detection of limbal epithelial stem cell deficiency. The aim of this study was to evaluate a novel combination of limbal stem cells (LSCs) maintained on contact lenses (CLs) in the presence of a 3T3 feeder cell layer regarding preservation of stem cell phenotype and the potential use for future in vivo transplantation. Methods Limbal epithelial cells were isolated from rabbit cornea and cultured with 3T3 cells on CLs. The preservation of LSC phenotype was determined using p63α and ABCG2 immunostaining, whereas epithelial differentiation was evaluated using CK3 and CK19. The colony-forming assay was used to determine the percentage of LSCs in cultures. Finally, CLs seeded with PKH26-labeled LSCs were transferred to rabbit eyes after performing a surgical keratectomy, and the transition and phenotype of labeled cells on the corneal surface were evaluated in whole-mount corneas. Results Proliferation of individual limbal cells was observed on CLs with a 3T3 feeder cell layer, showing holoclone formation and retention of viable stem or progenitor cell phenotype. Finally, a higher transition of cultivated cells after a dual sequential CL transplantation to the ocular surface was observed, showing the preservation of the LSC phenotype in the corneal surface. Conclusions Limbal cells cultivated on a CL carrier overlaying a 3T3 feeder layer are mitotically active and retain the LSC phenotype. This novel technique of using CLs as a carrier offers an easily manipulable and nonimmunogenic method for transferring LSCs for ocular surface reconstruction in patients with limbal epithelial stem cell deficiency.

1 The morphological consequences of hypobaric hypox ia, exposure to reduced pressure atmospheres, were examined in the hippocampus of male Fischer 344 rats. Severe chronic hypoxia can produce permanent neuronal damage with hippocampal structures being especially vulnerable. 2 Hippocampal morphology was studied using histolo gical observations after a 4 day exposure to sea level, 5500 m, or 6400 m. Two groups tested at 6400 m were sacrificed at different intervals following exposure, 72 and 144 h, to examine the effect of post-exposure time on neuronal damage. 3 Histological damage was observed in rats' brains following exposure to altitude, with cell degeneration and death increasing as altitude increased. In addition, it was found that the longer the time following exposure before sacrifice, the more noticeable the damage, suggest ing delayed neurotoxicity. Increases in the number of damaged cells following altitude were significant for the CA3 region of one 6400 m group; however, other differ ences did not reach statistical significance. Rats exposed to altitude for 4 days ate less and lost significantly more weight than did animals at sea level. 4 It appears that 4 days of exposure to altitudes less than or equal to 6400 m does produce changes in the CA3 subfield, but the damage is different than that seen with other models of non-transient ischemia.

PURPOSE Ocular injuries after exposure to the vesicant sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed limbal stem cell deficiency (LSCD), expressed clinically by corneal neovascularization and epithelial defects. The present study aimed to investigate the involvement of corneal nerves in the development of the delayed LSCD. METHODS Rabbit eyes were exposed to SM vapor and observed clinically up to 1 month. Morphology and density of corneal nerves were studied in acetylcholinesterase-stained whole-mount corneas at different time points after exposure. Corneal calcitonin gene-related peptide (CGRP) was measured and the relation to clinical symptoms was tested. RESULTS Degeneration of nerve terminals was observed a few hours after exposure simultaneously with the typical signs of SM ocular toxicity. Although corneal erosions healed within days, the nerves continued to disintegrate under a Wallerian degeneration pattern and their density declined significantly at 1 week in both central and peripheral corneal regions. Sprouting and regenerative nerve fibers were observed later in most of the corneas; however, healing was partial and often abnormal and was correlated with corneal edema. CGRP levels decreased at 24 hours and then increased significantly at 1 to 4 weeks, concomitant with the reinnervation process and development of the late injuries. CONCLUSIONS The prolonged impairment of corneal nerves, together with chronic inflammation implied by edema, and abnormal increase in CGRP may contribute to a pathological environment for corneal epithelial stem cells, leading to their death and to the development of the SM-induced delayed LSCD.

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.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.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.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.

The hippocampus processes memory is an early target of aging-related biological and structural lesions, leading to memory decline. With absent neurodegeneration in the hippocampus, which identified in rodent model of normal aging the pathology underlying age-related memory impairment is not complete. The effective glial–vascular networks are the key for maintaining neuronal functions. The changes of glial cells and cerebral capillaries with age may contribute to memory decline. Thus we examined age associated changes in neurons, glial phenotypes and microvasculature in the hippocampus of aged rats with memory decline. Young adult (6 months) and aged (35 months) male rats (Fisher/Norway-Brown) were used. To evaluate memory, four days of acquisition phase of Morris water maze tasks were carried out in both age groups and followed by a probe trial 2 h after the acquisition. The brains were then collected for analysis using immunochemistry. The aged rats showed a delayed latency (p

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.

The main objective of this research was to study the relationship between glucocorticoids, aging and the deterioration of cognitive functions. Towards this end, an attempt was made to develop an animal model which will enable the investigation of such interactions, using subcutaneously implanted sustained-release corticosterone pellets. The goal was to achieve moderately high concentrations of corticosterone in plasma, comparable to the peak basal levels or to those found under mild stress. Middle-aged (12 months old) Fischer-344 rats, used in this study, were divided before the prolonged hormonal treatment into cognitively ‘impaired’ and ‘non-impaired’ groups using the Morris water maze. The cognitive impairment, which was induced by the long-term corticosterone administration, was exhibited during acquisition of the 8-arm radial maze only in the ‘non-impaired’ group. Behavioral scores for drug-treated ‘impaired’ rats were not statistically different from those of the placebo-treated ‘impaired’ group. The morphological deterioration in hippocampal areas of the brain was quantified and revealed high correlation with the behavioral data. This animal model may become extremely useful in testing projected prophylactic therapy against the brain damage and cognitive deficits induced by the high corticosteroid-aging combination.

Stress was implied as involved in "enhanced aging," and prolonged administration of corticosterone was claimed to lead to central neuronal lesions. This study describes an animal model that simulates the steroid elevation associated with stress by a continuous slow-release administration of corticosterone, in young (3 months old) and middle-aged (12 months old) Fischer 344 rats. Plasma concentrations of corticosterone were stable throughout the day, with no diurnal variation, within the range associated with mild stress. Corticosterone prolonged treatment resulted in morphological changes mainly in the CA1, CA4, and dentate gyrus areas of the hippocampus. Middle-aged rats showed higher vulnerability to the long-term COR treatment than young ones, even when COR treatment was prolonged in young rats from 63 to 90 days. Middle-aged rats were screened before the corticosterone treatment, using the Morris water maze, and divided between cognitively "impaired" and "nonimpaired" subpopulations. Severe cognitive damage during acquisition of the eight-arm radial maze was shown, after the continuous hormonal treatment, in rats initially defined as "nonimpaired" in the Morris water maze. This animal model might be useful for testing the protective effects of drugs against brain changes and cognitive damage, during either pathological or normal aging.

The efficiency of the transdermal penetration of physostigmine through the guinea-pig skin, in a vehicle containing propionic acid, was studied in the course of developing a transdermal physostigmine pad for the potential treatment of Alzheimer's disease patients. In an attempt to improve drug penetration, the addition of various concentrations of oleic acid(0.5–5%) was tested. The study included histological examination of skin biopsies from application site. The addition of 0.5% oleic acid to the vehicle enhanced the permeation of physostigmine and shortened the time needed to reach steady state levels of blood cholinesterase inhibition, without any damage to the skin. Stable values of physostigmine concentration and of cholinesterase inhibition were measured both in blood and in brain up to 72 h. A high correlation was found between physostigmine concentration in the plasma and cholinesterase inhibition in whole blood samples (r = 0.99 for the values measured up to 48 h). A lower correlation was found between physostigmine concentration and cholinesterase inhibition in the brain (r = 0.57). In a vehicle containing up to 1% oleic acid, physostigmine was stable for at least one month. Oleic acid concentrations higher than 1%, while increasing the flux of the drug through the skin, resulted in considerable skin damage. © Wiley-Liss, Inc.

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.

Experiments were performed with Mongolian gerbils to study the effect of the specific nitric oxide (NO) synthase inhibitor N G - nitro- l -arginine ( l -NNA) on ischemic brain damage induced by 5 min bilateral carotid occlusion. A single i.p. injection of l -NNA did not result in any neuronal loss in the central nervous system. In animals undergoing ischemia, a selective destruction of hippocampal CA1 cells was observed whereas pretreatment with 50 mg/kg l -NNA 4 h before administration of ischemia produced significantly more extensive cell damage in the hippocampus and other brain regions. These findings demonstrate that in this model inhibition of nitric oxide generation augments ischemia-induced neuronal cell injury in the brain.

The long-term histopathological effects of acute lethal (95 μg kg-1) and sublethal (56 μg kg-1) doses of soman were studied in rats and were compared to lesions caused by equipotent doses of either another cholinesterase (ChE) inhibitor, DFP (1.8 mg kg-1), or a non-organophosphorus convulsant, metrazol (100 mg kg-1). Severe toxic signs were noted following one LD50 dose administration of all the compounds, yet only soman induced brain lesions. Moreover, even when administered at a sublethal dose (0.5 LD50), soman induced some histological changes without any clinical signs of intoxication. Soman-induced brain lesions were assessed quantitatively using a computerized image analyser. The analysis was carried out for up to 3 months following administration, and a dynamic pattern of pathology was shown. The cortical thickness and area of CA1 and CA3 cells declined significantly as early as 1 week post-exposure. No pathological findings were detected following DFP and metrazol administration. It is therefore suggested that brain lesions are not common for all ChE inhibitors and that convulsions per se are not the only factor leading to brain damage following the administration of soman. The degenerative process (found also with the sublethal dose of soman) might be due to a secondary effect, unrelated to soman's clinical toxicity, but leading to long-term brain injuries.