Comprehensive Modeling of Corneal Alkali Injury in the Rat Eye.

PURPOSE: To characterize the molecular, clinical, and histopathological profiles in the rat cornea after alkali injury over a 21-day period. METHODS: Alkali injury was induced in one eye of male Lewis rats. Corneal opacity and corneal neovascularization were assessed daily. Real-time qRT-PCR analysis and immunohistochemical staining were conducted to examine inflammation, neovascularization, and fibrosis. RESULTS: We found that within 2 hours of chemical exposure, corneal opacification rapidly developed with an acute increase in various cytokine expressions, while several cytokines demonstrated a secondary peak by day 7. Early neutrophil infiltration peaked at day 1 post-injury while macrophage infiltration peaked at day 7. Throughout the time course of the study, corneal opacity persisted and neovascularization, lymphangiogenesis, and fibrosis progressed. CONCLUSIONS: This study highlights the molecular, clinical, and histopathological changes throughout the progression of alkali injury in the rat cornea. These profiles will assist in the development of new strategies and therapies for ocular alkali injury.

Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts.

BACKGROUND: Angiogenin (ANG), a component of tears, is involved in the innate immune system and is related with inflammatory disease. We investigated whether ANG has an immune modulatory function in human corneal fibroblasts (HCFs). METHODS: HCFs were cultured from excised corneal tissues. The gene or protein expression levels of interleukin (IL)-1beta (ß), IL-4, IL-6, IL-8, IL-10, complements, toll-like receptor (TLR)4, myeloid differentiation primary response gene (MYD)88, TANK-binding kinase (TBK)1, IkappaB kinase-epsilon (IKK-ε) and nuclear factor-kappaB (NF-κB) were analyzed with or without ANG treatment in tumor necrosis factor-alpha (TNF-α)- or lipopolysaccharide (LPS)-induced inflammatory HCFs by real-time polymerase chain reaction (PCR), Western blotting and immunocytochemistry. Inflammatory cytokine profiles with or without ANG were evaluated through immunodot blot analysis in inflammatory HCFs. Corneal neovascularization and opacity in a rat model of corneal alkali burn were evaluated after application of ANG eye drops. RESULTS: ANG decreased the mRNA levels of IL-1ß, IL-6, IL-8, TNF-α receptor (TNFR)1, 2, TLR4, MYD88, and complement components except for C1r and C1s and elevated the mRNA expression of IL-4 and IL-10. Increased signal intensity of IL-6, IL-8 and monocyte chemotactic protein (MCP)-1 and MCP-2 induced by TNF-α or LPS was weakened by ANG treatment. ANG reduced the protein levels of IKK-ε by either TNF-α and LPS, and decreased TBK1 production induced by TNF-α, but not induced by LPS. The expression of NF-κB in the nuclei was decreased after ANG treatment. ANG application lowered corneal neovascularization and opacity in rats compared to controls. CONCLUSION: These results demonstrate that ANG reduces the inflammatory response induced by TNF-α or LPS in HCFs through common suppression of IKK-ε-mediated activation of NF-κB. This may support the targeting of immune-mediated corneal inflammation by using ANG.

[Features of stress reactivity of the organism and clinical patterns of ocular burn].

Clinical patterns of ocular burn in patients with different types and variants of stress reactivity were studied. It is shown that patients with the maladaptive type of stress reactivity at any stage of the disease are at higher risk of complications. Rapid exhaustion of the sympathoadrenal system or a hyperactive stress response seen in cases of the labile and hyperergic individual adrenergic immune reactivity of the organism (IAIRO) can be considered as manifestations of a regulation disorder. The absence of complications along with mild changes in immune reactivity is characteristic of the normoergic variant of IAIRO.

Bone marrow-derived mesenchymal stem cells affect immunologic profiling of interleukin-17-secreting cells in a chemical burn mouse model.

PURPOSE: This study investigated interleukin (IL)-17-secreting cell involvement in sterile inflammation, and evaluated the effect of mesenchymal stem cells (MSCs) on IL-17-secreting cell immunologic profiling. METHODS: Twenty mice were sacrificed at time points of 6 hours, 1 day, 1 week, and 3 weeks (each group, n = 5) after the cornea was chemically injured with 0.5N NaOH; IL-17 changes in the cornea were evaluated using enzyme-linked immunosorbent assay. Further, IL-17 secreting cells were assessed in the cervical lymph nodes by a flow cytometer. Rat MSCs were applied intraperitoneally in a burn model (n = 10), IL-17-secreting T helper 17 (Th17) cell and non-Th17 cell changes were checked using a flow cytometer in both cornea and cervical lymph nodes at 1 week, and compared with those in the positive control (n = 10). RESULTS: IL-17 was highest in the cornea at 1 week, while, in the cervical lymph nodes, IL-17-secreting cells showed early increase at 6 hours, and maintained the increase through 1 day to 1 week, and levels returned to the basal level at 3 weeks. Specifically, the non-Th17 cells secreted IL-17 earlier than the Th17 cells. When the MSCs were applied, IL-17 secretion was reduced in CD3(+)CD4(-)CD8(-), CD3(+)CD4(+)CD8(-), and CD3(+) CD4(-)CD8(+) cells of the cervical lymph nodes by 53.7%, 43.8%, and 50.8%, respectively. However, in the cornea, IL-17 secretion of CD3(+)CD4(-)CD8(-) cells was completely blocked. CONCLUSIONS: The results indicated that both IL-17-secreting non-Th17 and Th17 cells were involved in the chemical burn model, and MSCs appeared to mainly modulate non-Th17 cells and also partially suppress the Th17 cells.

[Clinical and immunological features of stress reactivity in the dynamics of ocular burn].

A series of clinical and immunological studies revealed peculiarities of stress reactivity in the course of ocular burn. According to adrenergic receptor expression level in active T-lymphocytes and its trend in the dynamics of ocular burn 2 types and 7 variants of individual adrenergic immune reactivity were identified and a physiologically adequate variant of stress reactivity was determined.

Bone Marrow-derived Mesenchymal Stem Cells Affect Immunologic Profiling of Interleukin-17-secreting Cells in a Chemical Burn Mouse Model

PURPOSE: This study investigated interleukin (IL)-17-secreting cell involvement in sterile inflammation, and evaluated the effect of mesenchymal stem cells (MSCs) on IL-17-secreting cell immunologic profiling. METHODS: Twenty mice were sacrificed at time points of 6 hours, 1 day, 1 week, and 3 weeks (each group, n = 5) after the cornea was chemically injured with 0.5N NaOH; IL-17 changes in the cornea were evaluated using enzyme-linked immunosorbent assay. Further, IL-17 secreting cells were assessed in the cervical lymph nodes by a flow cytometer. Rat MSCs were applied intraperitoneally in a burn model (n = 10), IL-17-secreting T helper 17 (Th17) cell and non-Th17 cell changes were checked using a flow cytometer in both cornea and cervical lymph nodes at 1week, and compared with those in the positive control (n = 10). RESULTS: IL-17 was highest in the cornea at 1 week, while, in the cervical lymph nodes, IL-17-secreting cells showed early increase at 6 hours, and maintained the increase through 1 day to 1 week, and levels returned to the basal level at 3 weeks. Specifically, the non-Th17 cells secreted IL-17 earlier than the Th17 cells. When the MSCs were applied, IL-17 secretion was reduced in CD3(+)CD4(-)CD8(-), CD3(+)CD4(+)CD8(-), and CD3(+) CD4(-)CD8(+) cells of the cervical lymph nodes by 53.7%, 43.8%, and 50.8%, respectively. However, in the cornea, IL-17 secretion of CD3(+)CD4(-)CD8(-) cells was completely blocked. CONCLUSIONS: The results indicated that both IL-17-secreting non-Th17 and Th17 cells were involved in the chemical burn model, and MSCs appeared to mainly modulate non-Th17 cells and also partially suppress the Th17 cells.

[Features of the stress-induced immunoreactivity of the organism in dynamics of the clinical current of the burn disease of eyes].

The individual adrenergical immunological reactivity of the organism (IA IRO) was investigated in 142 patients with eye burn of various degree. 2 types of IA IRO have been allocated by level and orientation of T-adrenoreception dynamics changes.

Bovine lactoferrin promotes corneal wound healing and suppresses IL-1 expression in alkali wounded mouse cornea.

PURPOSE: Using an in vitro cell culture model, bovine lactoferrin (BLF) stimulates healing of alkali-induced human corneal epithelial wounds. The present study examined the efficacy of BLF in promoting healing of corneal injury in vivo and explored BLF modulation of interleukin-1 (IL-1) during wound healing. METHODS: Alkali injury was induced to BALB/c mice by exposure of the mouse cornea to a sodium hydroxide (NaOH)-soaked filter disc for 2 min. The corneal surface was irrigated after the injury with saline. Topical BLF in phosphate buffered saline (PBS) (10 µl, 62.5 µM), bovine serum albumin (BSA) (10 µl, 62.5 µM in PBS) or PBS only (10 µl) were applied three times daily to both the alkali-injured and uninjured eyes for 3 d. Wound healing was assessed using 0.1% fluorescein staining under slit lamp microscope. The corneas at 6 h, 24 h or 3 d post-injury and treatment were excised and examined histologically, homogenized corneal tissue was evaluated for expression of IL-1α and IL-1ß. RESULTS: After 6 h post-wounding and treatment no significant reduction of wound area was observed between treatments and infiltrating cells or IL-1 expression were not elevated in any group. By 24 h, BLF-treatment resulted in accelerated wound closure (100%) compared to PBS and BSA treatment (70% and 65%, respectively). BLF treatment reduced infiltrating cells compared to controls and no elevation of IL-1, whereas controls displayed elevated infiltrating cells and increased levels of IL-1. After 3 d, mice treated with BLF exhibited complete wound closure while control corneas still exhibited some minor defects. Resolution of inflammation with minimal remaining infiltrating cells was observed in all corneas by day 3, coincident to normal levels of IL-1α and IL-1ß. CONCLUSION: BLF accelerated healing of corneal alkali injury in BALB/c mice which was associated with suppression of IL-1 and reduced infiltrating cells.

Anti-retinal antibodies in serum of laser-treated rabbits.

PURPOSE: Retinal injuries that affect the photoreceptors and/or the retinal pigment epithelium (RPE) may result in the leakage of retinal proteins into the systemic circulation. This study was designed to determine whether an immune response is elicited after an acute retinal injury resulting in circulating anti-retinal antibodies in the serum. METHODS: Fifty laser burns of different grades (minimally visible lesion [MVL], grade II [GII], or grade III [GIII] lesions) were created in the retinas of Dutch Belted rabbits. The degree of laser burns was confirmed by fundus imaging and histology. Serum samples were collected from the animals 3 months after the retinal injury. Candidate autoantigens were identified by two-dimensional (2-D) Western blots of rabbit retinal lysate probed with sera from either control or laser-treated animals. Candidate autoantigens were further characterized by immunostaining to confirm their retinal localization. RESULTS: Seven and 11 protein spots were selected from the MVL and GII laser-treated samples, respectively, for autoantigen identification. No protein spots were detected in the GIII laser-treated samples. Four candidate autoantigens were common to both MVL and GII lesions: dihydropyrimidinase-related protein 2, fructose-bisphosphate aldolase C, chaperonin-containing T-complex polypeptide 1 subunit zeta, and pyruvate kinase isozyme. CONCLUSIONS: Laser-induced retinal injuries resulted in circulating anti-retinal antibodies that were detectable 3 months after the injury. The response appeared to vary with the severity of the laser retinal damage. The identification of the candidate antigens in this study suggest that this approach may permit future development of new diagnostic methods for retinal injuries.

[Limbal stem cell deficiency after chemical burns : investigations on the epithelial phenotype and inflammation status]. / Limbale Stammzellinsuffizienz nach Verätzung : Untersuchungen zum epithelialen Phänotyp und Entzündungsstatus.

Limbal stem cell deficiency (LSCD) is clinically characterized by growth of fibrovascular pannus onto the corneal surface, chronic inflammation and impaired visual acuity. The aim of this study was to characterize the pannus tissue. Total RNA was isolated from six pannus samples and protein from three pannus samples from patients with LSCD caused by chemical burns. Normal human corneal tissue (n=6) and conjunctiva (n=6) were used as control tissues. The expression of the epithelial lineage markers keratin 3 (K3), K19 and MUC5AC, the inflammatory markers IL-1beta, ICAM-1 and VEGF was analyzed by Western Blotting and/or real-time PCR. Normal corneal tissue had a higher expression of K3 and a lower expression of K19 and MUC5AC in comparison to normal conjunctiva. A higher expression of inflammatory markers was seen in conjunctiva compared to corneal tissue. The pannus tissue showed a similar expression of lineage markers to conjunctiva but a higher expression of most inflammatory markers as compared to both control tissues. The analyzed samples of pannus in LSCD resembled conjunctiva and not cornea and showed a high expression of inflammatory markers indicating chronic inflammation.

Abnormal keratocytes and stromal inflammation in chronic phase of severe ocular surface diseases with stem cell deficiency.

BACKGROUND/AIMS: Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and alkali burns are associated with chronic, severe inflammation of the ocular surface that occasionally lead to corneal stem cell deficiencies. The corneal stroma in these diseases has not been studied comprehensively. The purpose of this study was to determine whether the keratocytes in the stroma were normal and whether the stroma remained inflamed in the chronic phase of these diseases. METHODS: Five pathological corneas, two with SJS, two with OCP and one with an alkali burn were examined. Corneal specimens were obtained during lamellar keratoplasty and the histological sections were immunostained with antibodies against CD34 and several cell surface antigens. The level of expression of proteoglycans (lumican, keratocan, biglycan) and chemokines (monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP) 1alpha, MIP1beta) were examined by quantitative real-time RT-PCR. RESULTS: The number of CD34-positive cells in the stroma was decreased and the expression level of biglycan increased in all of the pathological corneas. The numbers of CD45-positive and CD14-positive cells were increased in four of the five pathological corneas. The expression level of MIP1alpha and MIP1beta were markedly increased in all of the pathological corneas. CONCLUSIONS: These findings indicate that the keratocytes are abnormal and inflammation is still present in the corneal stroma in the chronic phase of SJS, OCP and alkali burns.

Local thermal injury elicits immediate dynamic behavioural responses by corneal Langerhans cells.

Langerhans cells (LCs) represent a special subset of immature dendritic cells (DCs) that reside in epithelial tissues at the environmental interfaces. Although dynamic interactions of mature DCs with T cells have been visualized in lymph nodes, the cellular behaviours linked with the surveillance of tissues for pathogenic signals, an important function of immature DCs, remain unknown. To visualize LCs in situ, bone marrow cells from C57BL/6 mice expressing the enhanced green fluorescent protein (EGFP) transgene were transplanted into syngeneic wild-type recipients. Motile activities of EGFP(+) corneal LCs in intact organ cultures were then recorded by time lapse two-photon microscopy. At baseline, corneal LCs exhibited a unique motion, termed dendrite surveillance extension and retraction cycling habitude (dSEARCH), characterized by rhythmic extension and retraction of their dendritic processes through intercellular spaces between epithelial cells. Upon pinpoint injury produced by infrared laser, LCs showed augmented dSEARCH and amoeba-like lateral movement. Interleukin (IL)-1 receptor antagonist completely abrogated both injury-associated changes, suggesting roles for IL-1. In the absence of injury, exogenous IL-1 caused a transient increase in dSEARCH without provoking lateral migration, whereas tumour necrosis factor-alpha induced both changes. Our results demonstrate rapid cytokine-mediated behavioural responses by LCs to local tissue injury, providing new insights into the biology of LCs.

[Immunomorphological study of time course of repair of corneal deep burn defects after stromal equivalent transplantation].

Early defects due to the afflux of monocytic phagocytes into the inflammatory focus, rapid resorption of detritis and normalisation of correlation between contents of matrix proteinases and their inhibitors, stimulation of stromal cornea keratinocytes migration and their proliferation characterize repair of postburn defects in transplantation of the stromal equivalent. Repair against the background of stromal equivalent transplantation is of a histotypical character: collagen gel organizes and serves the basis for migration of the cornea fibroblasts and temporary matrix for growing new epithelium, while restored basal membrane is a derivative of cornea epithelium.

[The parameters of the body immune reactivity in applying the therapeutic keratoplasty for the treatment of ocular burn lesions of extra severity]. / Pokazateli immunologicheskoi reaktivnosti organizma pri primenenii lechebnoi keratoplastiki v lechenii ozhogovoi bolezni glaz osobo tiazheloi stepeni.

One hundred and two (128 eyes) patients with stage 4 ocular burns underwent examinations. One-stage therapeutic keratoplasty was used in 67 (67.7%) patients. Retransplantation was made in 35 (34.3%) of patients due to graft rejection. According to the study, the more severe the clinical course of burn lesions is (cases that necessitated transplantation in the present research), the more pronounced changes are observed in the body immune reactivity of victims. In particular, the below peculiarities were observed: a progressing reduction of the contents of lymphocytes' and of B-lymphocytes; and a diminishing phagocytic activity of neutrophils in the peripheral blood registered at the terminal treatment stage in the patients after retransplantation versus the 1st-group patients with a more favorable clinical course. The compensatory increase of T-lymphocytes, T-helpers and of IgA, IgG was found to be less pronounced in group 2. A notably more intense sensitization response of the body to corneal antigens was established in the patients after retransplantation, which was a basis for using this index as a prognostication test in respect to the graft rejection crisis.

[Effects of immunization on proteinase-inhibitor balance in the lacrimal fluid and blood serum in alkaline burns of the cornea (an experimental study)]. / Vliianie immunizatsii na proteinazno-ingibitornyi balans v sleznoi zhidkosti i syvorotke krovi pri shchelochnom ozhoge rogovitsy (éksperimental'noe issledovanie).

Proteinase-inhibitor balance in the lacrimal fluid in eye burns was studied in rabbits with modified and intact immune status. Stimulation of the immune system was induced by complete Freund's adjuvant. Clinical picture of the disease was studied in parallel. Immunization accelerated the local adaptive and defense reaction of the proteolytic enzymes and their inhibitors, which was paralleled by a more benign course of burn disease of the eyes, in comparison with that in non-immunized animals.

Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury.

Previously, thymosin beta 4 (Tbeta(4)) was found to promote wound healing in full thickness skin wounds and heptanol debrided corneas. Here, the effect of Tbeta(4) was examined treatment on corneal wound healing and inflammation in vivo after alkali injury, a more severe wound of the eye. Corneas from 129 Sv mice were chemically burned with a 2 mm disc soaked in 1 N NaOH for 30 sec. Eyes were irrigated copiously with phosphate buffered saline (PBS) and then treated topically with either Tbeta(4) (5 microg/5 microl PBS) or 5 microl PBS twice daily. Animals were killed, the eyes were enucleated, fixed and embedded in plastic resin or prepared for mRNA analysis. Mouse corneas topically treated with 5 microg of Tbeta(4) twice daily after alkali injury demonstrated accelerated re-epithelialization at all time points and decreased polymorphonuclear leukocyte (PMN) infiltration at 7 days post injury (p.i.) when compared to PBS-treated controls. mRNA transcript levels were decreased several fold for interleukin (IL)-lbeta, and the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2 and monocyte chemoattractant protein (MCP)-1 from 1 to 7 days after injury in the Tbeta(4)- vs. PBS-treated corneas. Thus, Tbeta(4) may provide a new clinical treatment for severe traumatic corneal wound disorders by promoting rapid corneal wound healing and decreasing both PMN infiltration and inflammatory cytokine and chemokine mRNA levels.

[Immunologic experimental studies on the alkali burn of cornea in rats].

OBJECTIVE: To investigate the immune reaction of severe corneal alkali burns. METHODS: The models of severe corneal alkali burns were induced in rats. Immunohistochemistry was carried out on wholemounts of the cornea and iris at different time points after corneal burns to detect T-lymphocytes and their subpopulation, macrophages, dendritic cells and MHC class II -positive cells. RESULTS: In the early stage of the burns, there were small increasing number of T-lymphocytes and MHC class II-positive cells in the cornea and iris. At the period of corneal melting and perforation, a massive influx of lymphocytes (CD(+)(3), CD(+)(4), CD(+)(8)), macrophages and dendritic cells was seen in the cornea adjacent to the limbus and iris. CD(+)(4) lymphocytes were much more than CD(+)(8) in number. T-cells and MHC class II-positive cells were observed not only at the limbus but also in the melting area and corneal center. Morphological changes of these cells were also noted. In the restoration stage, a number of positive cells were still found in the iris. CONCLUSION: Immunological mechanism is involved in the pathological changes of the damage induced by corneal alkali burns, especially in the pathogenesis of the corneal melting and perforation.

[Natural cytokine complex as a drug bioregulator in healing of alkaline burns of cornea (experimental morphological study)]. / Estestvennyi kompleks tsitokinov kak lekarstvennyi bioreguliator pri zazhivlenii shchelochnykh ozhogov rogovitsy (éksperimental'no-morfologicheskoe issledovanie).

Clinical morphological features of reparative processes of alkaline burns of the cornea are studied in 24 rabbits. Effects of the natural cytokine complex on this process are validated. Burn process is regarded as an immune inflammation. Morphological and morphometric data demonstrate the mechanism of the cytokine effect on the reparative processes in the cornea, based on the regulation of the inflammatory postburn and proliferative processes. The repair of the cornea after burn in experiment and control is described in detail.

[Immunohistochemistry studies on the thermal burn of cornea in rats].

PURPOSE: To investigate immunological mechanism of corneal melting perforation and inflammation after thermal burn. METHODS: The models of severe corneal burns were induced with thermal metal in rats. Immunohistochemistry was carried out on wholemounts of the cornea, iris, choroid-sclera comples, retina at different time point after corneal burns, to derect T-lymphocytes and their subpopulation, macrophages, dendritic cells, MHC class II-positive cells. RESULTS: In the early stage of the burns, there were small number of increasing T-lymphocytes, MHC class II-positive cells in the cornea and iris. At the period of corneal melting and perforation, a massive influx of lymphocytes (CD3, CD4, CD8), macrophages and dentritic cells were seen in the cornea adjacent to limbers and iris. CD4 positive cells were much more than CD8 in number obviously. MHC class II-positive cell were found not only at the limbus but also in the melting area and corneal centre. Morphological changes of these cells were also noted. At restoration stage positive cells decreased gradually. CONCLUSIONS: Immunological mechanism is involved in the pathogenesis of the damage induced with corneal thermal burns, especially in the pathogenesis of corneal melting and perforation after burn.