Pediatric Carbon Monoxide Poisoning in Southern Israel: A Cross-Sectional Study.

OBJECTIVES: This study aimed to describe the epidemiology and clinical burden of unintended carbon monoxide (CO) poisoning among children in the Negev region of southern Israel. METHODS: This was a cross-sectional retrospective study of CO poisoning patients admitted to Soroka University Medical Center in 2011 through 2015. RESULTS: Overall, 43 cases of CO poisoning were recorded among children younger than 18 years. Five patients died, all upon admission. Poisoning due to smoke "per se" and due to CO emitted from heating devices were responsible for 28 (65.1%) and 14 (32.6%) cases, respectively. Eight (18.6%) patients suffered from convulsions, and 13 (43.3%) of 30 evaluable patients complained of headaches. Twenty-two (51.2%) were found unconscious in the field, and 7 (16.3%) were unconscious at examination at the emergency department. The average carboxyhemoglobin level on admission was 10.5% ± 10.4% (level ranging from 0.1% to 46.2%). Treatment included oxygen in 34 patients (79%) and hyperbaric oxygen therapy in 8 patients (19%). No differences were found between Bedouin and Jewish children in sex, age, residence area, source of CO poisoning, symptoms severity, and need for hyperbaric oxygen therapy. More patients with exposure to water heating devices were older than 4 years, lived in villages, and were diagnosed as having loss of consciousness in the field compared with those exposed to smoke inhalation. CONCLUSIONS: Carbon monoxide poisoning in children is frequent in southern Israel. Education about prevention, implementation of safer standards for home heating systems, and government supervision are required management strategies to decrease the CO poisoning incidence in southern Israel.

A comprehensive analysis of corneal mRNA levels during sulfur mustard induced ocular late pathology in the rabbit model using RNA sequencing.

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 naïve 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 naïve 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 naïve 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 naïve 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.

Whole body exposure of rats to sulfur mustard vapor.

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.

Successful single treatment with ziv-aflibercept for existing corneal neovascularization following ocular chemical insult in the rabbit model.

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.

Differential expression of corneal and limbal cytokines and chemokines throughout the clinical course of sulfur mustard induced ocular injury in the rabbit model.

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-1ß, 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 naïve eyes. At 1w, corneal IL-1ß, IL-6, IL-8 and TNFα and limbal IL-8 and MCP-1 levels were significantly higher compared to naïve eyes. During the late pathology, at 4w, elevated levels of corneal IL-1ß, 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-1ß 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.

Intratesticular and incisional line infiltration with ropivacaine for castration in medetomidine-butorphanol-midazolam sedated dogs.

OBJECTIVE: To evaluate whether intratesticular and incisional ropivacaine infiltration produces sufficient intra- and postoperative analgesia for castrating dogs under sedation. STUDY DESIGN: Randomized, blinded, controlled clinical study. ANIMALS: Twenty-three healthy dogs weighing 5.8-35.6 kg admitted for castration. METHODS: Dogs were sedated with medetomidine (0.01 mg kg-1), butorphanol (0.2 mg kg-1) and midazolam (0.2 mg kg-1) intramuscularly, and were randomly assigned to group R, 0.2-0.4 mL kg-1 of ropivacaine 0.5%, or group S, an equivalent volume of saline injected intratesticularly and along the incision line. If persistent motion was observed during surgery, sedation was considered to be insufficient and general anaesthesia was induced. Carprofen 2.2 mg kg-1 was administered postoperatively. Pain was evaluated in all dogs before sedation and postoperatively following atipamezole administration at 1, 2, 4, 8 and 24 hours using an interactive visual analogue scale (IVAS; 0-100), the Glasgow composite pain scale-short form (CMPS-SF; 0-24), and a mechanical algometer. Methadone 0.3 mg kg-1 was administered intravenously to dogs if IVAS >30 or CMPS-SF >4. RESULTS: There was no significant difference between groups for the number of dogs administered general anaesthesia. The time from the beginning of surgery to induction of general anaesthesia was significantly shorter [median (range)] in group S [6 (3-25) minutes] than in group R [56 (36-76) minutes]. At 8 hours IVAS was significantly higher in group S (14 ± 10) than in group R (6 ± 4). CONCLUSIONS AND CLINICAL RELEVANCE: Intratesticular and incisional ropivacaine infiltration delayed the time to anaesthesia induction, and provided analgesia after castration performed under deep sedation in dogs. Intratesticular local anaesthesia can be an important part of the anaesthetic plan for castration.

Acute and long-term ocular effects of acrolein vapor on the eyes and potential therapies.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Comparison of structural, architectural and mechanical aspects of cellular and acellular bone in two teleost fish.

The histological diversity of the skeletal tissues of fishes is impressive compared with that of other vertebrate groups, yet our understanding of the functional consequences of this diversity is limited. In particular, although it has been known since the mid-1800s that a large number of fish species possess acellular bones, the mechanical advantages and consequences of this structural characteristic - and therefore the nature of the evolution of this feature - remain unclear. Although several studies have examined the material properties of fish bone, these have used a variety of techniques and there have been no direct contrasts of acellular and cellular bone. We report on a comparison of the structural and mechanical properties of the ribs and opercula between two freshwater fish - the common carp Cyprinus carpio (a fish with cellular bone) and the tilapia Oreochromis aureus (a fish with acellular bone). We used light microscopy to show that the bones in both fish species exhibit poor blood supply and possess discrete tissue zones, with visible layering suggesting differences in the underlying collagen architecture. We performed identical micromechanical testing protocols on samples of the two bone types to determine the mechanical properties of the bone material of opercula and ribs. Our data support the consensus of literature values, indicating that Young's moduli of cellular and acellular bones are in the same range, and lower than Young's moduli of the bones of mammals and birds. Despite these similarities in mechanical properties between the bone tissues of the fish species tested here, cellular bone had significantly lower mineral content than acellular bone; furthermore, the percentage ash content and bone mineral density values (derived from micro-CT scans) show that the bone of these fishes is less mineralized than amniote bone. Although we cannot generalize from our data to the numerous remaining teleost species, the results presented here suggest that while cellular and acellular fish bone may perform similarly from a mechanical standpoint, there are previously unappreciated differences in the structure and composition of these bone types.

Normograde and retrograde pinning of the distal fragment in feline humeral fractures.

OBJECTIVE: To determine if normograde and retrograde pinning of the distal humeral fragment, performed to maximize pin purchase in this fragment, would damage vital structures in and around the elbow and shoulder joints in cats. STUDY DESIGN: Anatomic study. SAMPLE POPULATION: Cadaveric cats (n = 12; 24 thoracic limbs). METHODS: Twelve thoracic limb pairs were harvested from domestic short-haired cats and 1 limb from each pair was allocated to 1 of 3 groups. A transverse osteotomy was created at the junction of the middle and distal thirds in the diaphyseal osteotomy group (n = 8) and proximal to the supracondylar foramen in the metaphyseal osteotomy group (n = 8). Humeri in the normograde group (n = 8) were left intact. Retrograde pinning of the distal fragment in both osteotomy groups was performed with the elbow flexed. Pins were driven into the medial epicondyle until they exited the skin caudal to the elbow and dissection of the soft tissues around the exit tract of the pin was performed. The fracture was reduced and the pin was advanced until it exited the proximal humeral fragment. In the specimens in normograde group, pinning was initiated on the distal aspect of the medial epicondyle. A 1.0 mm guide hole was drilled from the medial epicondyle to the center of the medullary cavity of the distal humeral metaphysis. A bevelled 1.5 mm IM pin was driven proximally through the guide hole until it exited the proximal humerus. RESULTS: Pins exiting the distal aspect of the medial epicondyle passed through muscle origins in 5 specimens in the diaphyseal osteotomy group and in all 8 specimens in the metaphyseal group. The ulnar nerve was entrapped in 1 specimen in both the metaphyseal osteotomy and diaphyseal osteotomy groups. The articular cartilage of the trochlea was damaged in 5 specimens in the diaphyseal osteotomy group and in 1 specimen in the metaphyseal osteotomy group. There was no damage to articular or periarticular structures by pins exiting the proximal humerus. CONCLUSION: Retrograde pinning of the distal fragment in humeral fractures in the cat may damage the articular cartilage and cannot be recommended. Normograde pinning is safe and maximizes pin purchase in the distal fragment.

Bilateral ureteral obstruction in a cat due to a ureteral transitional cell carcinoma.

A 15-year-old cat was presented with a history of lethargy and vomiting. Serum biochemistry revealed severe azotemia. Ultrasonography revealed a small left kidney and hydronephrosis of the right kidney. There was an abdominal mass between the kidneys. Necropsy revealed a mass circumflexing both ureters and histopathology confirmed a diagnosis of transitional cell carcinoma.

The Effect of Increasing Tibial Tuberosity Advancement and Quadriceps Muscle Force on Cranial Translation of the Tibia in the Cranial Cruciate Deficient Stifle Joint in Dogs.

Background: Cranial cruciate ligament (CCL) disease is a well-known pathology that generates both rotational and translational instability of the stifle joint that leads to osteoarthritis in dogs. Tibial tuberosity advancement (TTA) is a common surgical technique used to dynamically neutralize the tibiofemoral shear forces to achieve stifle joint stability. However, significant persistent instability has been documented in clinical cases. The purpose of this study was to evaluate the effect of increasing quadriceps load, increasing tibial tuberosity advancement, and increasing joint flexion angle on the cranial translation of the tibia relative to the femur in the cranial cruciate ligament deficient stifle joint. Methods and Results: Six cadaveric hind limbs were collected from six healthy mixed breed dogs of medium build and prepared for biomechanical testing. The specimen was placed into a custom-made joint testing machine, and translation of the tibia relative to the femur was measured at stifle angles of 135°, 120°, and 105°. Cranial tibial thrust was generated by applying a vertical load to the metatarsal pad and the quadriceps muscle was simulated with loads of 0, 5, and 10 kg applied to the patella via a system of weights and pulleys. All specimens were tested with the CCL intact and cut, both of which served as controls. The tibial tuberosity was then advanced using both 6 mm and 9 mm cages, and the specimen was tested using the identical technique. Each specimen was loaded to failure by increasing the load applied to the pes until the sudden marked cranial translation of the tibia. Tibial tuberosity advancement with an applied quadriceps load was successful in limiting cranial tibial translation in 54/62 tests. Under similar loading conditions, TTA failed to limit cranial translation in 8 tests. The failures occurred at all angles of flexion examined. In the cases that failed cranial translation could be limited by either increasing the quadriceps load or increasing the amount of tibial tuberosity advancement. Conclusion: This study showed that TTA with an applied quadriceps load is effective at decreasing cranial tibial translation at functional joint angles.

Ocular Surface Changes After Sulfur Mustard Exposure in Rabbits, Monitored by Impression Cytology.

PURPOSE: Ocular injuries after exposure to sulfur mustard (SM) are characterized by acute corneal erosion and inflammation of the anterior segment that may be followed by delayed corneal neovascularization and epithelial defects, associated with limbal stem cell deficiency in part of the exposed eyes. This study aimed to further clarify the mechanism of the late injury by monitoring SM-induced cytological alterations in the ocular surface, in relation to the clinical symptoms, using impression cytology (IC). METHODS: Rabbit eyes were exposed to SM vapor (n = 20) and were clinically observed up to 4 weeks. Samples for IC were collected simultaneously from the upper bulbar conjunctiva, limbus, and cornea and then fixed and stained with periodic acid-Schiff and hematoxylin. At 1 month, animals were killed and eyes dissected and processed for histology. RESULTS: Concomitant with clinical symptoms of SM ocular toxicity, IC showed significant long-term loss of conjunctival goblet cells shortly after exposure, followed by abnormal differentiation toward squamous metaplasia. Simultaneously with corneal erosion, apoptotic bodies and cellular debris were seen in the corneal epithelium, followed by regeneration at 1 week. Migration of conjunctival goblet cells toward the cornea was noted in neovascularized eyes, as early as 1 week, indicating limbal stem cell deficiency. The IC findings were supported by histological evaluation. CONCLUSIONS: Continuous monitoring of the ocular surface after SM exposure by IC enables earlier detection of pathology and therapeutic intervention, therefore, is recommended for routine follow-up of casualties. Prolonged loss of goblet cells may point toward the role of mucin in the pathogenesis.

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.

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 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.

The beneficial effects of doxycycline, an inhibitor of matrix metalloproteinases, on sulfur mustard-induced ocular pathologies depend on the injury stage.

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.

Intrathoracic pseudocyst in a kitten.

A 2-month-old, intact, female domestic shorthair kitten presented with a history of acute-onset dyspnoea. Severe dyspnoea and tachypnoea were noted on physical examination. Serosanguinous fluid, consistent with a modified transudate, was aspirated from the pleural cavity immediately after the physical examination, with an immediate decrease in respiratory rate and effort. The thorax was radiographed and the entire left hemithorax appeared to be filled with a large soft tissue density mass. Thoracic ultrasound was performed and a cystic structure, measuring 3.0 cm × 1.5 cm, was seen in the left hemithorax. An explorative thoracotomy was performed and a mass obliterating the left hemithorax was found. The mass was removed by a combination of blunt and sharp dissection. A final diagnosis of thoracic pseudocyst was made on histological examination of the tissue. The mass was described as a sterile process characteristic of an organised seroma or haematoma. Recovery from surgery was uneventful and the kitten was discharged 48 h postoperatively. The kitten was still alive with no recurrence of clinical signs at the time of writing this report, 8 months postoperatively.

Prolonged impairment of corneal innervation after exposure to sulfur mustard and its relation to the development of delayed limbal stem cell deficiency.

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.

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.