Science topic

Retinal Degeneration - Science topic

A retrogressive pathological change in the retina, focal or generalized, caused by genetic defects, inflammation, trauma, vascular disease, or aging. Degeneration affecting predominantly the macula lutea of the retina is MACULAR DEGENERATION. (Newell, Ophthalmology: Principles and Concepts, 7th ed, p304)
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I am working with mouse retinal samples which are lysed in RIPA buffer. I was looking to use lamda protein phosphatase but the components of RIPA inhibit the phosphatase. Any recommendations on a phosphatase to use? I will be running western blots on these samples.
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I think it is unlikely that any added enzyme will retain much activity in RIPA buffer, which is denaturing, unless it is an exceptionally stable enzyme.
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I am doing immunofluorescence staining on retinal sections from RCS rats to assess GFAP. As shown in my attached image, the GFAP staining is not consistent with most literature I've seen with retinal GFAP. Typically, I see long connected processes throughout the length of the retina following injury. But as shown in my image, I get short processes. If anyone has any information on if I am doing something wrong or explanation about the images, I would greatly appreciate help.
Here are all the details that might help:
  • RCS rats are a retinal degeneration model, they were p90 when euthanized
  • fixed with Davidson's (formaldehyde based), paraffin embedded, cut at around 7-10 microns (I think 7)
  • did heat antigen retrieval using citrate buffer at pH 6 (I know I probably don't need to but I have to for a co-stain I will use in the future)
  • blocking buffer is 5% goat serum and 0.1% triton-X in PBS, GFAP antibody is rabbit polyclonal (dako/agilent z0334) diluted 1:1000 in blocking buffer (I did a titration all look similar in terms of structure of GFAP), and secondary is goat anti-rabbit conjugated with alexa fluor 594 (invitrogen, A-11037 ) diluted 1:500 in blocking buffer
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Very useful to know. Many thanks Kabir
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IT is a picture of the fundus. I am not sure about what is this indicative of and what landmarks should I take into consideration. I have acquired a lot of such images but I am not able to interpret them.
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Indeed - it is the holangiotic fundus of an albino rat or mouse. The nerve head and retinal vessels look normal - what are the clinical findings of the animal - is there any change in vision or behaviour?
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optical coherence tomography (OCT) is used with considerable success to study retinal degeneration. I am trying to evaluate the status of biomarker discovery of neuro-retinal degeneration in Macular Degeneration using OCT and related imaging systems
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The potential of using OCT especially, swept source and OCTA for AMD may provide more markers that can predict macular degeneration.
This review covers the topic quite well. The potential of spectral domain optical coherence tomography imaging based retinal biomarkers. Int J Retina Vitreous. 2017; 3: 1.
Published online 2017 Jan 9. doi: 10.1186/s40942-016-0054-7
Additional sources:
1.de Sisternes L, Simon N, Tibshirani R, Leng T, Rubin DL. Quantitative SD-OCT imaging biomarkers as indicators of age-related macular degeneration progression predicting AMD progression using SD-OCT features. Invest Ophthalmol Vis Sci. 2014;55:7093–7103. doi: 10.1167/iovs.14-14918. [PubMed] [Cross Ref]
2. Wu Z, Luu CD, Ayton LN, Goh JK, Lucci LM, Hubbard WC, Hageman JL, Hageman GS, Guymer RH. Optical coherence tomography-defined changes preceding the development of drusen-associated atrophy in age-related macular degeneration. Ophthalmology. 2014;121:2415–2422. doi: 10.1016/j.ophtha.2014.06.034. [PubMed] [Cross Ref]
3. Ouyang Y, Heussen FM, Hariri A, Keane PA, Sadda SR. Optical coherence tomography-based observation of the natural history of drusenoid lesion in eyes with dry age-related macular degeneration. Ophthalmology. 2013;120:2656–2665. doi: 10.1016/j.ophtha.2013.05.029. [PMC free article] [PubMed] [Cross Ref]
4.Choroidal Changes Associated With Subretinal Drusenoid Deposits in Age-related Macular Degeneration Using Swept-source Optical Coherence Tomography.
Laíns I, et al. Am J Ophthalmol. 2017.
5.Consensus Definition for Atrophy Associated with Age-Related Macular Degeneration on OCT
November 2017Ophthalmology
DOI10.1016/j.ophtha.2017.09.028
I hope they can contribute to the solution of your question.
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My research is related to cell line study in diabetics and retinopathy, does anyone know of any animal activity labs that do these kinds of experiments?
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Yes sundar ---  Animal model - In vitro culture system and in vivo animal model ( mice, rat and rabbits) have been applied to find the intraretinal microvascular abnormalities
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retinitis pigmentosa is the most common inherited human eye disease resulting in night blindness and visual defects.
as a biomedical engineer i like to know how can we help this patient by using ips cells.
thanks for sharing your information with me.
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Dear Maede,
Yes you can model the human disease, as previously shown for other inherited diseases such as ALS (motoneuron disease). Using cells, such as skin cells, from normal and diseased individuals, you can generate/induce pluripotent stem cells. Then you can also use iPS cells to generate organoids to compare the properties of normal vs disease conditions. I have heard of some trials of stem cell treatments/transplantations of patients in retinal diseases but I am not sure about the long term outcomes.
best wishes, Refik
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I want to induce retinal degeneration in vitro in a specimen of goat's eye. Which chemical can be used for the purpose?
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The latest approach Intravitreal injection of sodium iodate best results with 0.8mg
Good luck !
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I am interested in transcorneal electrical stimulation and I'd like to start an experiment with this tecnique, but I am having troubles in finding mice corneal electrodes for this purpose.
Thanks in advance for your help!
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Dear Christian,
You will probably need to build your now and it is relatively easy. You will need to have a adjustable stimulator first of all. Then you will need 2 insulated metal wires (e.g., copper), one for (-) and one for (+) line, each 50 cm 1m depending on the distance. Twist the two wires together except in each end, remove the insulation at the tip where you will stimulate. You can adjust the distance between the two electrical poles according to the field of stimulation of cornea. The other side of the wires will go to the stimulator and will need to be fitted with a suitable pin that will connect to the stimulator unit. Although I used my electrodes to stimulate nerves or the brain parenchyma, basically it is the same principal. Examples of these stimulation units and electrodes you can see in my previous in vivo and in vitro publications, I will include a couple below. 
Best wishes, Refik
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Hi everybody,
Can anyone describe the exact protocol to set up this animal model of retinal degeneration?
My questions are as simple as:
    (i) When you put back to normoxia the mice at P12 (after the 5-days period of 75% hyperoxia), do you just take the cage outside the oxygen chamber back to the atmosphere normal room conditions or do you keep them in the cage and set up the oxygen rate to normoxia?".
   (ii) Could you provide me a list of the material I need to set up this model?
   (iii) Are there any trick I should know before starting?
   (iv) Are C57BL/6 sensitive to this treatment?
   (v) How do you manage the death of the feeder mother? I mean do you specially buy some feeder mice for each experiment or do you use some feeder mice from you animal facility?
Thanks a lot for all the answers to those questions and for all the détails you could bring into my knowledge!
Juliette
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I have found that it is potentially possible to use airtight cages for mice and perfuse them with oxygen under went in order to not fill the lab room with oxygen. I'm still looking for a cheap and good oxygen analyser for this work. 
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we are conducting a lab research to test a type of stem cells in the retinitis pigmentosa. I want to know which animal model is the most suitable for the research.
I know it may depend only upon the choice of the research team but I need to know which aspect should I look for in the model?like what would make the research process less problematic. What will give the same result that will be in human ? etc
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Hi,
There are two lines of mice deficient in the β-subunit of phosphodiesterase (rd1 and rd10 mice). It is easier to work with mice than dogs. There are a lot of papers using this aninañ model
In rd1 a degeneration of photoreceptors starting at P8 and progressing to complete loss of the rods by P20. it has several limitations. The massive and rapid degeneration of photoreceptors leaves only a narrow window for treatment. Additionally, the onset of cell death overlaps with the final differentiation of the retina, making it difficult to distinguish between abnormal development and degeneration.
However, the mutant line rd10 offers a longer therapeutic window. In rd10 mice, rods begin to degenerate between P16 and P20, with maximum cell death occurring between P21 and P25 in a central to peripheral gradient. By P60, rods are no longer detectable and only cones remain.
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I want to better understand this from the physical point of view. Thank you in advance!
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For which purpose?
In the case of lighteninig , the incoherent light does not damage the eye sensors (retina) because of low brightness and  wide spectral range, whereas the coherent light (laser) gives out  very intense brightness to be dangerous for eyes and other optical sensors even in the same optical power. Furthermore, the incoherent light emits wide divergence useful for lightening, conversely coherent light undergoes inherently much smaller divergence leading to directed energy purposes  to illuminate smaller areas in comparison.
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I have been using a procedure for mouse eye fixation in paraformaldehyde. We remove the mouse eye after sacrafice. We immediately put it in 4% pfa for 30 minutes. After that we punch a hole in the eye, we do not remove the cornea or lens. After fixing for 4 hours, we put it in 10% sucrose for an hour, 20% sucrose for an hour, then 30% sucrose overnight. We then put the fixed eye into a cryomold filled with OCT mixture without sucrose. We freeze the tissue on dry ice. Sometimes the retina is perfectly sectioned, other times, it is extremely detached and has cells missing or shrunken. Any advice would be helpful. Thanks
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Be very careful when taking the eyes out. Sometimes just a little too much pressure and the retina will detach and no matter what you do after it is too late.
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I am planning to do injections in mice with Metripranolol. Found that the drug is practically insoluble in water/saline. What do people using this drug use as a solvent when they do injections? Thanks in advance for your answers.
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Dear Dr. Kanan:
During my tests on rats using poorly water soluble drugs, I found very useful to dissolved compounds in DMSO 8%, but also using physical combinations methods such as ultrasonic disruption. To use DMSO will avoid possible side reactions to certain solvents in the animal tests. Best regards.
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I'm mostly interested in early life disorders, but late life disorders may also be of interest.
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This is probably a bit beside you point, but something I found quite interesting when I bumped into it: Visual Advantage in Deaf Adults Linked to Retinal Changes.
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I am doing western blotting for synaptic vesicle protein and it gives double band with smeared pattern (manufacturers details).
I am also getting the same pattern in wild type mouse but this specific pattern of bands is absent in retinal degeneration model. I am getting a single band. I am not sure how to compare the two. This specific protein is glycosylated in nature. Any suggestions?
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Changes in N-glycosylation of cone opsin proteins have been described in the RPE65 KO model of Leber's congenital amaurosis (severe retinal degeneration) by Sato et al., Exp Eye Res 2010.