Experimental study of VEGF immune expression dynamics in the retina using photoinduced BRVO model

Aim. To describe the dynamics of vascular endothelial growth factor (VEGF) immune expression in the retina using the model of photoinduced branch retinal vein occlusion (BRVO) and to establish the terms of neovascularization appearance.Materials and methods. BRVO was modelled on 21 chinchilla rabbits (21 eyes) weighing 1.5‑2 kg (fellow eyes served as controls). Photosensitizer «Fotoditazin» (2.5 mg / kg) was injected intravenously. 15 min later, transpupillary laser irradiation of branch retinal vein near the optic nerve head was performed. Irradiation energy density was 200 J / cm2. Histological analysis and immunohistochemistry of the retina was performed following 30 min, at days 1, 2, 3, 7, 14 and 30.Results. Maximum VEGF accumulation in photoinduced BRVO model was observed on day 2. From day 3, direct neovascularization was confirmed. VEGF levels were stably high throughout the follow-up to the day 30 inclusive.Conclusion. VEGF immune expression in the retina using the model of BRVO induced by photodynamic exposure was explored for the first time. These data can serve as the basis for future studies in order to define optimal anti-VEGF agent, its dosage and terms to manage this condition

The neuronal insulin sensitizer dicholine succinate reduces stress-induced depressive traits and memory deficit: possible role of insulin-like growth factor 2.

BACKGROUND: A number of epidemiological studies have established a link between insulin resistance and the prevalence of depression. The occurrence of depression was found to precede the onset of diabetes and was hypothesized to be associated with inherited inter-related insufficiency of the peripheral and central insulin receptors. Recently, dicholine succinate, a sensitizer of the neuronal insulin receptor, was shown to stimulate insulin-dependent H2O2 production of the mitochondrial respiratory chain leading to an enhancement of insulin receptor autophosphorylation in neurons. As such, this mechanism can be a novel target for the elevation of insulin signaling. RESULTS: Administration of DS (25 mg/kg/day, intraperitoneal) in CD1 mice for 7 days prior to the onset of stress procedure, diminished manifestations of anhedonia defined in a sucrose test and behavioral despair in the forced swim test. Treatment with dicholine succinate reduced the anxiety scores of stressed mice in the dark/light box paradigm, precluded stress-induced decreases of long-term contextual memory in the step-down avoidance test and hippocampal gene expression of IGF2. CONCLUSIONS: Our data suggest that dicholine succinate has an antidepressant-like effect, which might be mediated via the up-regulation of hippocampal expression of IGF2, and implicate the neuronal insulin receptor in the pathogenesis of stress-induced depressive syndrome.journal articleresearch support, non-u.s. gov't2012 Sep 182012 09 18importe

Димерный дипептидный миметик 1-й петли фактора роста нервов ГК-6, активирующий PI3K/AKT и МЕК/MAPK/ERK, вызывает дифференцировку клеток РС12 по нейрональному типу

Dimeric dipeptide mimetic 1st loop of NGF GK-6 (10-6M) activating as phosphatidylinozitol 3-kinase/Akt and mitogen-activated protein kinase MEK/MAPK/ERK) signaling cascades causes PC12 cell differentiation into neuron-like cells. At the same time dimeric dipeptide mimetic the 4th loop of NGF GK-2 (10-6M), which activates only phosphatidylinozitol 3-kinase/Akt, does not possess the differentiating effect.Димерный дипептидный миметик 1-й петли NGF ГК-6 (10-6М), активирующий как фосфатидилинозитол-З/АИ-киназный, так и митоген-активируемый протеинкиназный (MEK/MAPK/ERK) сигнальные каскады, вызывает дифференцировку клеток РС12 по нейрональному типу. В то же время димерный дипептидный миметик 4-й петли NGF ГК-2 (10-6М), активирующий только фос-фатидилинозитол-3/Akt- киназный путь, не обладает дифференцирующим действием

LOCAL SUBRETINAL INJECTION TECHNIQUE OF XENOGENIC STEM CELLS LABELLED BY MAGNETIC PARTICLES IN EXPERIMENT

Purpose. To develop a technique for local subretinal injection of xenogeneic stem cells labeled with magnetic particles and to prove experimentally its effectiveness.Material and methods. We used a line of stem cells HEK-293 GFP, labeled with magnetic particles. The study was made in 84 eyes of 42 chinchilla rabbits aged 6 months, the weight was from 2.5 to 3.5kg. All right eyes were experimental (42 eyes) and all left eyes (42 eyes) were in the control group. In the experimental group we used original complex of polymer elastic magnetic implant (PEMI) with a laser probe and fixed it to the sclera, then we made a median vitrectomy and injected HEK-293 GFP under the retina using a specially designed dispenser. In the control group PEMI was not fixed. We examined animals using biomicroscopy, ophthalmoscopy, ultrasound scanning, optical coherence tomography (OCT), computer tomography (CT), morphological study (cryo-histological sections) 1, 3, 5, 7, 14 days and 1 month after surgery.Results. According to the results of biomicroscopy in observation periods up to 3 days the vascular injection was visualized in the area operation. According to the results of ophthalmoscopy and ultrasound scanning during the first day the local retinal detachment was visualized in the area of local injection of the stem cells, which was not noted in terms of further observations. CT helped us to confirm the localization of PEMI fixation. The morphological study results showed that cells were located in the subretinal space up to 14 days in the experimental group, and only up 3 days in the control group.Conclusion. The developed surgical technique enables to control the injection of cells into the subretinal space, reduces the risk of tissue damage and exit cells in the vitreous space. The suggested method allows to fix the cellular material in the local place of the injection and enables to predict cells` movement

Development and experimental basis of local subretinal technique of xenogenic’s injection stem cells labelled by magnetic perticles

Purpose: is to develop a technique for local subretinal injection of xenogeneic stem cells labeled with magnetic particles and to prove experimentally its effectiveness.Material and methods: We used a line of stem cells HEK-293 GFP,labeled with magnetic particles. The study was made on 84 eyes of 42 chinchilla rabbits 6 months of age, the weight were from 2.5 to 3.5 kg. All right eyes were experimental (42 eyes) and all left eyes (42 eyes) were the control group. In the experimental group we used original complex of polymer elastic magnetic implant (PEMI) with laser probe and fixed it to the sclera, then we made a median vitrectomy and injected HEK-293 GFP under the retina using a specially designed dispenser. In the control group PEMI was not fixed. We examined animals using biomicroscopy, ophthalmoscopy, ultrasound scanning, optical coherence tomography  OCT), computer tomography (CT), morphological study (cryohistological sections) in 1, 3, 5, 7, 14 day and 1 month after surgery.Results: According the results of biomicroscopy in observation periods up to 3 days the vascular injection was visualized in the area operation. According the results of ophthalmoscopy and ultrasound scanning in 1 day the local retinal detachment was visualized in the area of local injection of the stem cells, which was not visualized in terms of further observations. CT helped us to confirm the local place of PEMI fixation. The morphological study results showed that cells were located in the subretinal space up to 14 days in the experimental group, and only up 3 days in the control group.Conclusion: The suggested surgical technique enables to control the injection of cells into the subretinal space, reduces the risk of tissue damage and exit cells in the vitreous space. The suggested methodology allows the fixing of the cellular material in the local place of the injection and enables to predict cells`s movement

Using of magnetic particles for fi xing of isolated cells in subretinal transplantation

Purpose: This study focuses on the development of the method of introduction of magnetic microparticles in the cytoplasm of HEK-293 cell line with their subsequent fixation under the retina of the eye.Materials and Methods. Magnetic particles (d = 2,8 mm) were treated with pluronic and injected into the cytoplasm of HEK-293 cell line, expressing GFP. The surgery was made under general anesthesia. HEK-293 containing magnetic particles were injected into the subretinal space of rabbit eyes (eyes 96, 48 rabbits) using original dosing device. In the experimental group (48 eyes, 24 rabbits) we fixed episcleral magnetic implant to hold cells in local place. In the control group (48 eyes, 24 rabbit) magnetic implant was not fixed. After the surgery all animals were examined using biomicroscopy, ophthalmoscopy with photographic recording, ultrasound, computed tomography and morphological study in certain terms (1, 3, 5, 7, 14, 21 day and 1 month).Results: The introduction of the magnetic particles into the cytoplasm of HEK 293 cell line has no effect on cell viability. HEK-293 containing magnetic particles remains in the place of injection during 21 days in rabbit eyes, where the magnetic implants were fixed (in control group during 3 days). Conclusions: Using of cells containing magnetic particles with fixation of the magnetic implant can be a promising method for cell therapy for the treatment of retinal diseases

PATHOGENETIC MECHANISMS OF EPITHELIALIZATION AND FORMATION OF PERSISTENT ULCERS OF THE GRAFT IN PATIENTS WITH DESTRUCTIVE CORNEAL PROCESSES AFTER PENETRATING KERATOPLASTY

Keratoplasty in patients with recurrent destructive processes in the cornea nowadays remains one of the most serious problems in ophthalmic surgery.Purpose. The analysis of results of penetrating keratoplasty (PK) in patients with destructive processes of the cornea or the corneal graft depending on the initial level of GDNF and the activity of the MAP kinase cascade of the signaling pathways.Material  and  methods.  The  analysis  of  the  results  of  PK  was performed in 23 patients (34 eyes) with acute destructive processes of cornea or the corneal graft in different etiologies. The follow-up period was from 6 months to 2 years. The analysis of the results of treatment was carried out depending on the condition of the corneal graft at various times after PK.The  immunohistochemical  examination  of  the  biological  material (corneal disks of the recipient) was performed using antibodies to GDNF, phospho-ERK1 / 2, phospho-JNK1 / 2, Ki67, GAP43, Bcl2 and Bax.Results.  The  time  of  epithelialization  of  the  graft  after  PK  in destructive processes of the cornea was increased in 79% of cases. When the epithelialization of the corneal graft was completed more than 6 and 10 days after PK in the postoperative period persistent erosions of the graft (PEG) with ulceration were formed in 22% and 89% of cases, respectively.The intensity of immunohistochemical reactions with antibodies to GDNF, phospho-ERK1 / 2, phospho-JNK1 / 2 in corneal disks at the epithelialization time of up to 5, 10 days and more eythan 10 days after PK was reduced from moderately positive to weakly positive and negative, respectively.When the time of epithelialization was completed in 6-10 days and there was not PECG, the intensity of reactions with antibodies to GDNF remains moderately positive, phospho-ERK1 / 2, phospho-JNK1 / 2 was lightly positive; when there was PEG the intensity of all reactions was weakly positive.Conclusion. The severity of the destructive pathological process in the cornea and postoperative complications after PK in patients with destructive corneal processes depend on the degree of GDNF deficiency, the decrease in the activity of the MAP kinase cascade of signaling pathways; the density of nerve plexuses in the own corneal tissue or corneal graft, and the activity of apoptosis processes