Annotation of the results obtained in 2018
INVESTIGATION OF APPROACHES TO IMPROVEMENT OF RETINAL PROTECTION AGAINST LIGHT-INDUCED DAMAGE In 2018, the feasibility of mitochondria-targeted antioxidant therapy for prevention of iatrogenic retinal photodamage was investigated. Intense illumination by visible light is known to be produced by a number of ophthalmic instruments. Therefore, the retina can be exposed to damaging irradiation doses during a wide range of ophthalmologic examinations and surgeries, especially those accompanied by a temporary displacement of the protective/refractive eye media. Furthermore, retinal photodamage can be induced not only by visible light, but also by UV irradiation, which is normally absorbed by the cornea and lens, but can reach the retina upon iatrogenic damage of these tissues (for example, during photorefractive surgery). A common feature of the light-induced retinal damage is photosensitized oxidative stress, which leads to degeneration of photoreceptors and other retinal neurons. Since the development of oxidative stress and apoptosis of retinal neurons involves mitochondria, a promising approach to the prevention of these processes is premedication using mitochondria-targeted antioxidants. In this project, pathological processes induced by UV and visible light were modeled in the retina of experimental animals (rabbits Oryctolagus cuniculus) and the efficacy of mitochondria-targeted antioxidant PDTP for prevention of retinal photodamage was characterized. The following results were obtained. Firstly, the maximum doses of irradiation of the patients‘ retina with various ophthalmological visible light sources were recalculated. Given these refined data, the iatrogenic retinal photodamage was stimulated by irradiation of the animals with 2200 lux or 30000 lux visible light for 3 hours yielding irradiation doses of 118 J/cm2 and 1620 J/cm2, respectively. It was demonstrated that, in the first case, irradiation does not cause acute or chronic damage to the retina. Meanwhile, in the second case, the irradiation promotes severe degenerative and compensatory changes in this tissue, including a massive loss of photoreceptors and other neurons via apoptosis, activation, vacuolization and migration of RPE cells, as well as retinal detachment and phagocytic activity of RPE. Thus, visible light irradiation at a dose of 1620 J/cm2 was chosen as the optimal procedure for modeling uncomplicated iatrogenic retinal photodamage. Using this model, the mechanism and dynamics of photodamage/regeneration of the retina in the absence of treatment were characterized. It was shown that apoptosis of photoreceptors is triggered already in the course of the irradiation. One day later apoptotic changes were also found in the neurons of the INL and GCL, whereas on the third day a massive loss of these cells was observed, as indicated by the decrease in the thickness of the respective nuclear layers. Neuronal death is accompanied by retinal detachment, as well as activation, migration and phagocytic activity of RPE cells. After seven days, these changes are followed by compensatory processes (activation of the Müller glial cells) and inflammation (granulocyte infiltration, increased number of fibroblasts in choroid, edema). The observed reduction of the ONL thickness at 1000 μm distance from the optic nerve disc (~20%) was recognized as an objective morphometric criterion for the retinal photodamage. According to electrophysiological studies, the irradiation causes a dramatic decrease of the functionality of photoreceptors and other retinal neurons and, after 7 days, leads to severe reduction of photosensitivity of this tissue. The described morphometric and electrophysiological criteria for retinal photodamage were applied for further characterization of the retinoprotective effect of PDTP (see below). It was shown that the irradiation is accompanied by pronounced oxidative stress of retinal cells, involving both their cytosolic components (H2O2 accumulation) and cellular lipids (increased MDA levels). The oxidative stress is spontaneously resolved only after 7 days of the post-exposure period. At the same time, the development of the stress affects the antioxidant protection of the retina, causing a dramatic increase in AOA and a decrease in the activity of antioxidant protection enzymes 1-3 days after the irradiation. Since these changes persist over the next 4 days, it was assumed that they reflect the regulated long-term response of retinal cells to irradiation/oxidative stress. The changes in the redox status of photoreceptor proteins recoverin and visual arrestin in the irradiated retina were determined by western blotting. To this end, polyclonal (monospecific) antibodies against recoverin and arrestin were obtained by immunization of animals with purified recombinant antigens and affinity purification of the immunoglobulins from hyperimmune sera. It was shown that within 24 h after the exposure, disulfide dimers of both proteins are accumulated in retina and their elevated concentration remains unchanged for 3-7 days, which reflects the development of oxidative stress in retinal photoreceptors. It was demonstrated for the first time that recoverin can be oxidized in vivo to form a sulfenic/sulfinic acid derivative. Thus, in the developed model of retinal iatrogenic photodamage the most pronounced pathological changes in this tissue were successfully induced. In addition, morphometric, electrophysiological and biochemical criteria for the development of pathological processes in the irradiated retina were formulated. The results were published in 2018 in the journals ANTIOXIDANTS (Q2), FRONTIERS IN MOLECULAR NEUROSCIENCE (Q1), BIOCHIMIE (Q1) and BIOCHEMISTRY (MOSCOW) (Q2). Using the developed model, the pronounced retinoprotective effect of premedication using the mitochondria-targeted antioxidant PDTP was demonstrated for the first time. The premedication was shown to prevent retinal atrophy, apoptosis of photoreceptors and other neurons, activation and phagocytic activity of RPE cells, as well as activation of Müller glia and inflammatory processes. Immediately after the irradiation and on the 1st day the retina is morphologically healthy, and its cytoarchitecture is fully preserved for the next 3-7 days: the average thickness of the ONL is not statistically different from the norm, which confirms the prevention of photoreceptor loss. It was established that premedication with PDTP almost completely sustains the normal electrophysiological activity of the retina, protecting both the photoreceptors and other neurons of this tissue. It was shown that the mechanisms underlying the retinoprotective effect of PDTP are directly related to its pronounced antioxidant activity, since premedication suppresses the upregulation of MDA and H2O2 and accelerates their recovery, produces a stimulating effect on AOA, prolongs the normal activity of GPx in the irradiated retina, as well as suppresses the formation of disulfide dimers of arrestin and recoverin in photoreceptor cells. Thus, it was demonstrated for the first time that the flooding of the animal retina with PDTP produces a pronounced protective effect, making the structure and function of this tissue resistant to photodamage. Based on the obtained results, as well as the available data on the safety and bioavailability of PDTP, we can recommen applying this pharmaceutical as premedication (six conjunctival installations of 7.5 μM PDTP every 10 minutes in the course of 1 hour) in patients prior to ophthalmic procedures associated with retinal irradiation with bright visible light. The results were published in 2018 in the journal ANTIOXIDANTS (Q2) and BIOCHEMISTRY (MOSCOW) (Q2). The additional studies performed in 2018 were devoted to the modeling of UV-induced retinal damage and investigating feasibility of antioxidant premedication using PDTP to prevent this kind of damage (this work was not announced in the original application). It was shown that morphological changes in the retina induced by UVB light (312 nm) resemble the changes produced by visible light (massive apoptosis of photoreceptors and other retinal neurons, nuclear layers atrophy, activation and phagocytic activity of RPE, retinal detachment and inflammatory response). However, in contrast to photodamage by visible light, which manifests focally, UV damage is more extensive and includes areas of total retinal atrophy. The high efficacy of the prophylactic use of PDTP with respect to prevention of UV-induced retinal damage was demonstrated for the first time. In this case, no patches of retinal detachment and RPE activation, and no signs of apoptotic, atrophic and inflammatory processes are observed. The retinoprotective action of PDTP was shown to be based on preventing UV-induced oxidative stress, as premedication inhibits MDA growth and sustains the normal activity of retinal GPx. In addition, the upregulation of AOA in the retina is observed in response to UV irradiation, in similar fashion as it was observed in the case of illumination with visible light. Taken together, these phenomena might reflect the common protective response of the retina to increased redox potential of the cellular environment caused by light emission of different wavelengths. Overall, it was demonstrated for the first time that PDTP prevents retinal damage caused by visible light and UV irradiation. In both cases antioxidant premedication using this compound produces similar effects on the retina based on the direct antioxidant action of PDTP. Taking into account the obtained data, we can recommend premedication (six conjunctival installations of 7.5 μM PDTP every 10 minutes in the course of 1 hour) with PDTP for patients for the prevention of UV damage associated with different ophthalmic operations (trans-PRK, surgical treatment of primary open-angle glaucoma). Taking into account extraordinary efficacy PDTP in corneal protection (see below), application of this pharmaceutical can provide comprehensive protection of eye tissues from UV damage. STIMULATION OF CORNEAL REGENERATION AFTER REFRACTIVE KERATECTOMY Finally, in 2018 we demonstrated high efficacy of PDTP in prevention of UV-induced damage to cornea upon when the antioxidant is used as premedication (these studies were not announced in the original application). Photorefractive keratectomy (PRK) is a common approach for correcting myopia by remodeling the corneal stroma with a UV laser. The operation has a number of complications due to the development of UV-induced oxidative stress and apoptosis of keratocytes, which aggravate the course of post-traumatic reaction and slow down corneal healing. Previously (please see Report-2017), we have demonstrated that irradiation of the cornea of experimental animals with 312 nm UV-light induces injuries (denudation of the basement membrane (complete absence of epithelium), apoptosis of keratocytes and endothelial cells), which structurally resemble corneal damage associated with PRK. Using this model, it was shown that postoperative application of PDTP prevents degeneration of stromal keratocytes and other corneal cells, improves epithelialization of the damaged tissue, accelerates the regeneration of its stroma, as well as significantly reduces the level of inflammatory infiltration and edema. The produced treatment is based on the ability of PDTP to suppress the oxidative stress of keratocytes and endotheliocytes that survived the UV damage, thereby possessing normal activity during tissue regeneration. At the same time, the efficacy of PDTP is limited due to the fact that it acts through the mitochondria of living cells, and their number after UV irradiation is significantly reduced. In this regard, it was suggested that PDTP could provide more pronounced benefit in respect to cornea when applied as premedication. In support of this suggestion, our clinical tests demonstrated that PDTP premedication almost completely prevented the development of UV-induced corneal erosions. According to histological analysis, the survival rate of epithelial cells and keratocytes under the conditions of UV irradiation is drastically improved and the development of local inflammation is suppressed. This outstanding benefit of PDTP is associated with its ability to effectively inhibit the development of UV-induced oxidative stress in the cornea, since the premedication with PDTP suppresses lipid peroxidation and prevents a decrease in AOA in response to UV radiation. It was suggested that, since medical manipulations (for example, transPRK) are usually scheduled in advance, UV damage to the cornea can be successfully prevented by premedication using PDTP. Taking into account the overall results obtained at this current stage of the Project, we can recommend intensive premedication with PDTP prior to various ophthalmologic operations with a risk of visible/UV light damage to both the retina and the cornea. Given the outstanding protective and therapeutic properties of PDTP revealed in the Project, it can be assumed that prophylactic and/or therapeutic use of this compound will provide a complex benefit for the eye tissues, accelerating their regeneration and ensuring their resistance to iatrogenic damage. The results were published in 2018 in the journal BMC OPHTHALMOLOGY (Q1), FEBS OPEN BIO (Q2) and BIOCHEMISTRY (MOSCOW) (Q2).

 

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Annotation of the results obtained in 2016
Prolonged general anesthesia, which is required in a wide spectrum of surgical procedures, is a common cause of iatrogenic corneal abrasions. According to literature, prevalence of this complication in surgical patients reaches 27-44%. The risk of vision deterioration lingers for several days into the post-narcosis period. It can manifest in a partial loss of field of vision and its acuity, accompanied by a pronounced pain syndrome, justified by an incredibly high density of corneal innervation. One of the main pathogenic factors contributing to perioperative corneal injury is anesthetic-induced decline in function of secretory glands that invokes changes in biochemical properties of the tear, resulting in a loss of its protective qualities. All this considered, the following problems have recently gained relevance: finding correlation between the time spent under general anesthesia and the development of corneal pathologies on clinical, morphological, and biochemical levels; characterization of the associated alterations in tear biochemistry; and, on the basis of these data, development of techniques for preventing perioperational corneal lesions, and therapeutic approaches to enhance tissue regeneration from the described damage. In order to find solutions to the specified problems, in year 2016 an experimental animal (rabbit, Orycrolagus cuniculus) model of corneal lesions, caused by prolonged (0,5 – 6 h) general anesthesia, was developed. According to the clinical studies, the prevalence of such complication in narcotized animals increases with time, from less than 10% for 60 min of anesthesia to 100% after 6-hour narcosis. These estimations agree with data, acquired for surgical patients. Furthermore, the severity of the damage varies from point lesions after 1-2 h of anesthesia to larger confluent lesions, involving a significant portion of eye surface, by the end of 6-hour narcosis. These data are supported by the results of the histological analysis, revealing first pathomorphological alterations in the cornea as early as after 1 h of general anesthesia and detecting development of medium to high degree corneal abrasions by the 3-6 hour of the narcosis. The alterations affect only the epithelial layer of the cornea (and are, correspondingly, defined as corneal abrasions), and involve the coagulation of flat epitheliocytes, followed by their desquamation after 1 h of anesthesia, larger focal injuries with hydropic dystrophy and desquamation of the exterior wing cells after 3 h of anesthesia, and even more severe diffusive abrasions with pronounced hydropic dystrophy of wing and basal cells and the development of apoptotic changes in all corneal layers by the 6 hour of narcosis. The developing abrasions are accompanied by alterations in biochemical properties of the cornea. For instance, more than 1 hour-long anesthesia leads to the onset of oxidative stress in the tissue while at the end of the 6-hour narcosis the level of lipid peroxidation becomes increased 10-fold. At the same time, no compensatory increase of innate antioxidant activity or the activity of major antioxidant defense enzymes in cornea is observed. These effects might contribute to the described above degenerative processes. Proteomic and biochemical composition of the pericorneal tear fluid is also affected by anesthesia. For instance, the observed alterations in total protein content in tears indicate anesthesia-induced disturbance in main lacrimal glands function. The proteomic analysis of the major tear proteins revealed that in the course of anesthesia serotransferrin and serum albumin levels are increased, whereas annexin A1 content declines suggesting that these proteins may be considered as potential markers of the investigated condition. Given the antioxidant properties of serotransferrin and serum albumin and anti-inflammatory activity of annexin A1, these alterations may imply the deterioration of the corresponding functions of tears. Indeed, the development of oxidative stress in cornea under general anesthesia could be associated with functional changes of the components of antioxidant defense, abundant in tears. This suggestion is supported by the observations, that as early as after 30-60 minutes after the onset of anesthesia, the total antioxidant activity, superoxide dismutase activity, and activity of proteins, involved in regulation of glutathione metabolism (glutathione peroxidase and glutathione reductase) are decreased. At the same time the activity of glutathione-S-transferase is elevated, which may be associated with its role in detoxification of the injected anesthetic. Additionally, the content of anti-inflammatory interleukins 4 and 10 declines in parallel with the increase in content of pro-inflammatory cytokines (namely tumor necrosis factor alpha and interleukin-6), pointing to existence of an inflammatory component in the pathogenesis of corneal abrasions. Clinical monitoring of the corneal regeneration during a 30-days post-narcosis period after 3-hour general anesthesia revealed that in 59% of cases the symptoms linger until the next day, in 56% of cases – for the following 3 days, and in 40% of cases they are sustained through the whole 7 days. Moreover, in the latter group the corneal damage tends to progress further, turning to chronicity. Yet, in general, the animals anesthetized for 3 and 6 h had 70% of corneal damage regenerated by the day 7 and 14, respectively. In tears, complete recovery of total antioxidant activity and activity of the antioxidant defense enzymes occurred in 3-7 days after the application of 3-6 h anesthesia. For most enzymes this recovery was followed by a compensatory increase of activity. Finally, the inflammatory response in tears regenerates in a somewhat delayed manner. For example the concentration of interleukin 10 is partially recovered after 7 days, while tumor necrosis factor alpha reaches its normal level after one month of the post-narcosis period. Using the developed model, the effectiveness of antioxidant or anti-inflammatory premedication/therapy techniques for prevention/treatment of the corneal abrasions, was tested. The results of clinical and histological studies indicate that conjunctival instillations of antioxidant PDTP have positive effect on corneal epithelium, whereas anti-inflammatory agent dexamethasone either does not improve the state of the disease or even aggravates it. Premedication with 7.5 μM PDTP was found to induce the most pronounced protective effect, with cornea reaching full recovery after 1 day from 6-hour anesthesia. It is consistent with the fact that instillations of the antioxidant in the same concentration (for both premedication and treatment) suppresses the development of oxidative stress in corneal cells and enhances total antioxidant activity, superoxide dismutase activity, and functioning of enzymes of glutathione metabolism in corneal extracts. Moreover, PDTP similarly affects the biochemical composition of tears, both accelerating the recovery of antioxidant protection (1 day post-narcosis) and providing evident anti-inflammatory effect, which includes elevated secretion of interleukins 4 and 10 soon after the post-anesthetic recovery. It should be mentioned that the revealed anti-inflammatory activity of PDTP may be considered for treatment of other inflammatory eye conditions such as uveitis. To summarize, on the current stage of the project, the experimental model of anesthesia-induced corneal abrasions is developed and characterized on clinical and morphological levels, and associated alterations in biochemical properties of cornea and precorneal tear film are revealed. It is found that the mechanism of this complication involves the development of oxidative stress in corneal cells, accompanied by decline in antioxidant protection of tears and onset of local inflammatory response. On the basis of these data, prophylactic and therapeutic measures, including local instillations of targeted antioxidant PDTP, are successfully tested. These measures prevent perioperative corneal abrasions and stimulate tissue regeneration, and therefore may be applied in clinical practice. It is also demonstrated, that preventive and therapeutic effects of PDTP are associated with suppression of oxidative stress in cornea, increasing its innate antioxidant status, as well as antioxidant and anti-inflammatory qualities of cornea protecting tear film. The results of this work are reported in 2 publications: 1. Zernii E.Yu., Golovastova M.O., Baksheeva V.E., Kabanova E.I., Ishutina I.E., Gancharova O.S., Gusev A.E., Savchenko M.S., Loboda A.P., Sotnikova L.F., Zamyatnin A.A. Jr., Philippov P.P., and Senin I.I. Alterations in tear biochemistry associated with postanesthetic chronic dry eye syndrome. Biochemistry (Moscow), 2016, Vol. 81, No. 12, pp. 1549-1557. http://protein.bio.msu.ru/biokhimiya/inpress/abs/BM16-243.html 2. Zernii E.Yu., Gancharova O.S., Ishutina I.E., Baksheeva V.E., Golovastova M.O., Kabanova E.I., Savchenko M.S., Serebryakova M.V., Sotnikova L.F., Zamyatnin A.A. Jr., Philippov P.P., Senin I.I. Mechanisms of perioperative corneal abrasions: alterations in tear film proteome. Biomeditsinskaya Khimiya, 2016, in press.

 

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Annotation of the results obtained in 2017
STIMULATION OF CORNEA REGENERATION AFTER REFRACTIVE KERATECTOMY Photorefractive keratectomy (PRK) is a common method of vision correction based on remodeling of corneal stroma using ultraviolet (UV) excimer laser. To access the stroma, on the first stage of PRK the corneal epithelium layer is removed either via mechanical scarification (classical PRK) or by laser ablation (transepithelial or trans-PRK). Regeneration of the cornea after the mechanical and UV damage plays a key role in patient rehabilitation process. The UV-damage of the cornea is associated with potential pathology as it is known to cause oxidative stress and apoptosis of keratocytes, what exacerbates post-traumatic reactions, postpones healing and can result in irreversible complications. In the project, both mechanical and UV-derived corneal injury was induced and characterized in rabbits (Oryctolagus cuniculus) and the obtained animal models were employed for trialing efficacy of mitochondria-targeted antioxidant therapy using plastoquinonyl-decyl-triphenylphosphonium bromide (PDTP) for prevention of corneal damage. The following results were obtained. (1) The dynamics of corneal regeneration after 50 and 100 µm-deep experimental trans-PRK was characterized on clinical level. It was found that the rate of corneal healing after 50 µm-deep trans-PRK corresponds to that after the common operations in patients (7 days), whereas deeper incision causes excessive ablation and induces complications, uncharacteristic for the standard operations. (2) The structure of corneal lesions in early post-operative period of trans-PRK was determined on histological level. It was found that ablation of corneal epithelium and stroma is accompanied by inflammatory infiltration and edema of the latter as well as apoptosis of stromal keratocytes, which might be associated with UV-induced oxidative stress. Regeneration of the cornea involves active re-epithelialization, keratocyte activation, and stromal extracellular matrix remodeling. The process takes at least 7 days, what is in agreement with the acquired clinical data and the pathological picture in patients undergoing similar surgeries. These results were used to design an experimental model of UV-induced corneal injury. (3) A model of corneal damage caused by UV radiation (UV-induced component of corneal injury associated with PRK) was developed. It was demonstrated that four-day 312 nm light irradiation of the cornea for 20 minutes per day triggers the development of lesions, which are structurally similar to those caused by excimer laser ablation. In particular, basal membrane denudation (complete loss of epithelium), stromal edema and apoptosis of keratocytes and corneal endothelium cells were observed. The stepwise mechanism of corneal regeneration was characterized. It involves epithelium and endothelium regrowth via active division of epitheliocytes on the wound edge, formation of re-epithelialization roll, and interaction of active epithelium with stroma. The latter is restored through activation of healthy keratocytes. Regeneration takes place simultaneously in all corneal layers and it becomes completed by day 7. It was emphasized that multiple apoptosis of stromal keratocytes can be associated with UV-induced oxidative stress, which manifested in the cornea as elevation of MDA content (the product of lipid peroxidation) in the tissue, and decrease of AOA and SOD (components of antioxidant defense), followed by their compensatory growth. It was suggested that corneal healing could be enhanced via oxidative stress suppression. It was concluded that the model accurately produces alterations in cornea associated with trans-PRK, what makes it feasible for studying complications of the operation and for testing of the appropriate therapeutic approaches. (4) The feasibility of PDTP based antioxidant therapy for enhancement of corneal regeneration after UV irradiation was demonstrated. It was shown that the most pronounced therapeutic effect is produced by conjunctival instillations of 7.5 µM PDTP, 1 drop 3 times per day. The therapy prevents the loss of stromal keratocytes and other corneal cells, facilitates re-epithelialization of damaged tissue area, enhances stromal remodeling, and reduces the level of inflammatory infiltrations and edema. In general, clinical and histological data indicate that the treatment accelerates corneal healing by 3 days. According to biochemical studies, the instillations of 7.5 µM PDTP also suppresses oxidative stress in the cornea without affecting compensatory increase of its intrinsic antioxidant defense mechanisms, what could underlie efficacy of the treatment. (5) А model of corneal injury following experimental scarification (mechanically induced component of corneal injury associated with PRK) was developed. It was found that mechanical removal of epithelium causes edema and inflammatory infiltration of the outer stroma, exudation in the anterior chamber, and keratocyte loss. At the same time, deeper stroma, Descemet’s membrane and endothelium are unaffected. The overall regeneration occurs faster than after UV irradiation and complete restoration of corneal surface is achieved by day 4 of the post-operative period. The biochemical studies reveal oxidative stress that manifested as elevation of MDA and decrease of AOA and SOD activity. In conclusion, the model simulates consequences of mechanical corneal injury produced on the first stage of classical PRK. These consequences can contribute to slower tissue regeneration and development of post-operational complications. (6) It was found that the application of antioxidant therapy involving conjunctival instillations of 7.5 µM PDTP accelerates corneal healing after experimental scarification by 1-2 days. In particular, the treatment restores faster normal clinical state of cornea, enhances its re-epithelialization and remodeling of its stroma, decreases inflammatory infiltration and exudation, and suppresses oxidative stress of its cells. It is suggested, that the antioxidant therapy is feasible for stimulation of corneal healing and prevention of post-operative morbidities after classic PRK involving tissue scarification. (7) The feasibility of antioxidant therapy involving conjunctival instillations of 7.5 µM PDTP for acceleration of corneal regeneration after experimental trans-PRK was demonstrated for the first time. It was shown that the treatment results in normalization of the clinical state of cornea by day 3 of post-operative period. These data allow recommending such treatment in all patients undergoing trans-PRK. (8) The analysis of the existing literature data revealed that stromal keratocytes play central role in corneal regeneration by participating in extracellular matrix remodeling and stimulating re-epithelialization via secretion of specific growth factors. Considering these findings together with the results obtained on the current stage of the project, it can be speculated that the observed therapeutic effect of PDTP is based on prevention of keratocyte apoptosis (and, possibly, improved survival of limbal stem cells) by effective suppression of oxidative stress, induced by UV radiation of the excimer laser. The detailed results of the analysis are described in the review article published in Current Medicinal Chemistry (WoS impact factor – 3,249). INVESTIGATION OF APPROACHES TO IMPROVEMENT OF RETINAL PROTECTION AGAINST LIGHT-INDUCED DAMAGE Retinal damage by visible light of high intensity is another common iatrogenic complication in modern ophthalmology. As this pathology involves photosensitized oxidative stress of the retina, the developed herein antioxidant therapy can be considered as a promising approach for prevention of such injuries. This strategy must be trialed using appropriate models of light-induced retinal damage. In order to develop such models, in 2017 the common doses of retinal irradiation imposed on patients from different ophthalmological devices were estimated. (1) The levels of retinal irradiation during ophthalmoscopy were measured. It was found that handheld slit lamps provide light power density of 0.01 W/cm2, while in the case of stationary slit lamps and ophthalmoscopes this value is in the range of 0.7-1 W/cm2. Meanwhile, the total light irradiation dose during a standard ophthalmoscopy examination can reach 900 J/cm2, which exceeds 3-fold the dose sufficient for photoreceptor loss and retinal pigment epithelium alterations. (2) The levels of retinal irradiation in the course of common ophthalmologic surgeries were measured/calculated. It was determined that the operative microscopes provide light density of 0.22-0.42 W/cm2. However, the dose received during surgical interventions would range between 504 J/cm2 (20 minutes-long operation) and 4536 J/cm2 (3 hours-long operation) and can be further increased by pre-operative ophthalmoscopy examinations. According to literature, the doses of 396 J/cm2 potentially cause photoreceptor loss, while the dose of 3800 J/cm2 can result in severe retinal damage. (3) It was calculated that the standard irradiation dose received during photodynamic therapy of subretinal neovascularization equals to 50 J/cm2. According to the literature, such irradiation could induce alterations in photoreceptor layer of the retina. (4) Based on the performed analysis, two potentially pathological irradiation doses, namely 900 J/cm2 and 4500 J/cm2, which are common in clinical practice, were selected. The effect of these doses on the retina of experimental animals will be investigated on the next stage of the project. PREVENTION AND TREATMENT OF CORNEAL DAMAGE CAUSED BY PROLONGED GENERAL ANESTHESIA (extension of 2016 report) (1) The results obtained on the first stage of the project (in 2016) were presented in four works published in 2016-2017 in Russian and international journals (including Oxidative Medicine and Cellular Longevity, WoS impact factor – 4,593). The results were also reported in four Russian and international meetings. The work presented in VII International conference «Problem of safety in anesthesiology» was included into the Top-10 of the best studies and was published in «Vestnik anesteziologii i reanimatologii» (RSCI – 0,303). (2) In 2017, the prospective practical application of the project results was demonstrated in clinical studies (previously unannounced work). In particular, the efficacy of the therapy employing 0.25 µM PDTP was demonstrated in a representative group of surgery patients underwent general anesthesia. It was found that the treatment leads to stabilization of their tear film and completely prevents development of corneal erosions in post-operative period. These data allows recommending such antioxidant therapy to all patients undergoing prolonged surgical interventions under general anesthesia.

 

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