Brain-derived neurotrophic factor on diabetic retinopathy in rats the impact of early neural
【Abstract】 Objective: To evaluate the intravitreous
injection of brain-derived neurotrophic factor (Brain derived neurotrophic factor, BDNF) before and after the STZ diabetic rats in tyrosine hydroxylase (tyrosine hydroxylase, TH) levels and dopamine can no longer sudden change in the number of cells. France: 9-week-old Wistar rats, STZ (streptozotocin,
STZ) intraperitoneal injection, made of diabetes model, in the
model building 2wk after intravitreal injection of BDNF in rat eye solution, in the model building 4wk after the execution of large mouse, remove the eye, take the retina and retinal organization Western blotting stretched immune staining was
observed in the TH levels in the retina, thus reflecting the retina of diabetic rats in the dopaminergic amacrine cell level changes. Results: The diabetic rat retina without injection of BDNF in TH protein levels decreased dopaminergic amacrine cell count and the gray-scale lower than the control
group were statistically significant. Experimental group was injected with BDNF in TH protein levels in retina, dopaminergic amacrine cell counts and the Gray and the control group no statistical difference. Conclusion: The early stage
of diabetes in the STZ diabetic rats, intravitreal injection of BDNF can increase the level of TH in the retina to enhance dopaminergic amacrine cell number.
【Key Words】 Wistar rats were diabetic retinopathy tyrosine hydroxylase brain-derived neurotrophic factor
Involvement of brain derived neurotrophic factor in early retinal neuropathy of diabetes in rats
Abstract AIM: To observe the changes of tyrosine
hydroxylase (TH) protein levels and the density of dopaminergic amacrine cells before and after the administering brain derived neurotrophic factor (BDNF) protein into the vitreous cavities of streptozotocin (STZ) reduced Wistar diabetic rats. METHODS : Adult male Wistar rats, 9 weeks of age, were injected intraperitoneally with STZ to create diabetes. At 2 week after the models were created, BDNF protein was administered into the vitreous cavities of rats. At 4 week after the models were created, the rats were killed
and the retina was removed for Western blotting and Whole-
mount immunohistochemistry for TH to observe the changes of TH and dopaminergic amacrine cells in retina. RESULTS: The protein levels of TH, the number of positive staining
dopaminergic amacrine cells and the staining gray scale of experimental group without BDNF were lower statistically. But there were no statistical differences between experimental group with BDNF and control group. CONCLUSION: In the early stage of STZ diabetic, administering BDNF protein into the vitreous cavities can increase TH protein levels and the density of dopaminergic amacrine cells in the STZ rats' retina.
* KEYWORDS: Wistar rats; diabetic retinopathy; tyrosine hydroxylase; brain derived neurotrophic factor
Traditional theory suggests that microvascular disease of diabetic retinopathy are, but in fact it is a kind of retinal neurodegenerative diseases. In humans and experimental animals, in diabetes shortly after, and in the vascular
complications prior to the molecular function of neurons had been changed. Diabetic neuropathy in the retina as early as vascular complications, and diabetes, has always been through a serious threat to patients with vision. Many studies have
shown that brain-derived neurotrophic factor (brain derived neurotrophic factor, BDNF) in retinal physiology and pathology has an important role in physiological processes [1,2], this paper reported in diabetic rat retina after intravitreal injection of BDNF in the casein acid hydroxylase (tyrosine hydroxylase, TH) levels and dopaminergic amacrine cells, changes in the number of early diabetic retinopathy to preliminary study the pathogenesis of neuropathy.
1 Materials and methods
1.1 Materials STZ (streptozotocin, STZ), the United States Sigma Corporation. TH immunohistochemical staining kit, Wuhan Boster Biological Co., Ltd.. BDNF freeze-dried powder,
the United States Upstate companies. Bovine serum albumin (BSA), USA Sigma Company. Balanced salt solution (BSS), the
U.S. company Alcon. China Medical University, Department of Laboratory Animal inbred 9wk healthy adult male Wistar rats age 60, body weight 180 ~ 220g, the eye through the slit lamp and ophthalmoscopy refractive interstitial clear, no fundus
lesions. Sub-cage farming, the standard grain feed, not
limited to water. Keeping a well-ventilated place at room
temperature for 18 ~ 25 ?, relative humidity 40% ~ 70%, 12h
light dark cycle. Urine dipstick, Guilin Sino-Hui Technology
Development Co., Ltd.. Blood glucose meters, blood glucose
test strips, Germany, Roche.
1.2.1STZ-induced diabetic rat model of citrate solution, 0.1mmol / L (pH4.5), high-pressure sterilization, STZ
concentration of 10g / L dissolved in which the use of pre-
preparation. Rats fasted water 12h, STZ 70mg/kg body weight intraperitoneally. Subsequently 48h, 72h, 1wk tail vein blood taken 3 consecutive fasting blood glucose measurements in order to fasting blood glucose is higher than 16.7mmol / L for modeling success, and the weekly monitoring of urine sugar 1
times, once every 2wk monitoring of blood glucose, the fasting blood glucose less than 16.7mmol / L were excluded.
1.2.2 Animal grouping and treatment in order to mold into a mouse as an experimental group and control group injected
the same dose of citrate salt solution (n = 30). Rats into the mold after 4wk, 100g / L chloral hydrate 3ml/kg body weight intraperitoneal injection of general anesthesia, the removal of bilateral eye back. The rats were killed cervical dislocation of the law.
1.2.3 BDNF was injected into the eye after injection of STZ or placebo intraperitoneally 2wk, every 3d injection of a second, a total of 5 times. BDNF lyophilized powder 1g / L soluble containing 1g / L bovine serum albumin (BSA) in
balanced salt solution (BSS) in the. 100g / L chloral hydrate
3mL/kg body weight intraperitoneal injection of general anesthesia, 20g / L of lidocaine after the success of a drop of topical anesthesia, rats were randomly selected side of the eye, using micro-injector to take the above-mentioned solution
was injected into rats 5μL the vitreous cavity, the
contralateral eye injected with the same dose of 1g / L BSA in BSS solution. Will appear vitreous hemorrhage, or eyeball atrophy being weeded out.
1.2.4 Western blotting detection of TH protein levels detected. The rats were placed under a microscope, the eye to the anterior segment, carefully isolated from the retina, the retina into 1mL EP tube, by adding lysis buffer, ice ultrasound, 4 ? centrifuged and the supernatant, -70 ? to
save under , pending inspection. Phenol reagent protein, on the kind of time to ensure that each sample the same amount of total protein. Rat anti-TH monoclonal antibody (1:100
dilution) as primary antibody, rabbit anti-mouse IgG antibody
(1:100 dilution) as secondary antibody. Application BandScan software analysis of the gray value of electrophoretic bands.
1.2.5 Retinal Immunohistochemistry stretched to the retina fixed, degreasing, in the 1g / L of PBS solution in the
hydration, the number of flap cut into the retina, internal limiting membrane face on the glass slide on the tile carefully. Specimens were put in with anti-TH monoclonal
antibody (1:100 dilution), 5g / L Triton X-100 in PBS solution
at 4 ? incubated 72h, PBS washed, home rabbit anti-mouse IgG
antibody (1:100 -fold dilution) at 4 ? incubated 24h, PBS
washed, DAB color. Neutral gum Fengpian, the home under the microscope. Could distinguish between dopaminergic amacrine cells. Shop for each piece count of five high power field in
the tag number of cells can be obtained the number of TH-
Statistical analysis: count data with mean ? standard
deviation () that, t test, using SPSS 12.0 data analysis package deal.
2.1 the experimental group and control group in the retina of the TH protein levels in TH protein levels with the same lane to standardize the level of β-actin can be seen
that the experimental group rats were injected BDNF is not
gray eyes TH protein was significantly lower than the
contralateral eye and the control group eyes P