Shlomit Dachir

Cognitive deficits induced in young rats by long-term corticosterone administration

Corticosterone slow-release pellets, implanted for 9 weeks in young Fischer 344 rats, resulted in continuous high plasma levels of the hormone which are comparable to those of rats under mild stress. One week following termination of the drug treatment, the rats were tested in an eight-arm radial maze. During the initial acquisition stages, corticosterone-treated rats exhibited cognitive impairments in contrast to placebo-treated rats. The deficits were observed in all three parameters which were monitored, the total number of errors, the number of correct entries out of the first eight, and the total time needed to complete the test. This study is the first to report specific behavioral decrements related to the previously observed morphological hippocampal changes induced by long-term corticosterone administration.

Sub-regional hippocampal vulnerability in various animal models leading to cognitive dysfunction.

Summary. 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.

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.

Potential anti-inflammatory treatments against cutaneous sulfur mustard injury using the mouse ear vesicant model.

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.

Inhalation exposure to sulfur mustard in the guinea pig model: clinical, biochemical and histopathological characterization of respiratory injuries.

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.

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.

AF64A Induced Cholinotoxicity: Behavioral and Biochemical Correlates

The postulated involvement of the cholinergic system in Alzheimer’s disease (AD) has highlighted the research and therapeutic approach of this affliction during the last decade (1). There is little doubt at the present stage that a clear hypofunction of the cholinergic system is in evidence in certain brain areas in AD patients. Other neurotransmitter systems seem to be relatively unaffected (1).

The Beneficial Effects of Doxycycline, An Inhibitor of Matrix Metalloproteinases, on Sulfur Mustard-Induced Ocular Pathologies Depend on the Injury Stage

Abstract Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

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.

Cultivation and characterization of limbal epithelial stem cells on contact lenses with a feeder layer: toward the treatment of limbal stem cell deficiency.

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 p63&agr; 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.