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Parkinson's Disease - Science topic

A topic to discuss Parkinson's disease research: Basic science, clinical research, treatment.
Questions related to Parkinson's Disease
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The PD patients I have known never smiled and showed little emotion on the face. Hence I came up with this hypothesis. If the answer is positive, it would be of clinical value.
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As a Movement Disorders specialist can say that facial expression is the point we assess within UPDRS scale, but it is not the only one feature. The diagnosis is made clinically on minimum 2 of 3 clinical signs (bradykinesia + rigidity and/or rest tremor). It seems that the error occurrence can be huge if only apply on AI based facial expression assessment. But may be when the physicians society will come to consensus about digital biomarkers on PD, we will have these features as the diagnostic one. But it seems to be a long process IMHO
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I am working of LUHMES cells lines. I differentiate them for live cell calcium imaging. On the day of imaging, I stained the with fluo4 in differentiating medium (pH=7.4) and incubate the cells at 37C.
I am acquiring images at 20x, time interval of 1s and sometimes 2s, exposure time (100-150ms) for 30 minutes.
After 5 minutes of acquisition, I stimulate them with 2uM of ATP.
When I analyse the images, I dont observe spikes, in the cells and all the cells behave differently. In most of the cells I dont observe oscillations but increasing concentration of calcium. I also try to do imaging in saline buffer and dpbs but apparently cells did not like it and were stressed even before starting imaging.
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Sundas Arshad Try adjusting the following factors one at a time to see which has the most positive effect on your imaging results.
1. Ensure that Fluo-4 is loaded effectively: Optimise loading time, dye concentration and wash steps to avoid excessive background fluorescence that could mask the calcium spikes.
2. The time resolution of 1-2 s may be too slow to capture fast calcium spikes, especially if they are transient. Reducing the interval to 0.5s or even faster may help to capture transient events. However, be aware of increased photobleaching and phototoxicity and find a balance that works for your cells.
3.ATP at 2 µM may not be sufficient to elicit a clear calcium response in all cells, especially if receptor sensitivity varies between cells; consider increasing the ATP concentration stepwise to determine the optimum level for consistent responses. Also ensure that receptors for ATP (such as P2Y or P2X receptors) are expressed equally on all cells and that their activity is influenced by the state of differentiation.
4. LUHMES cells, even differentiated ones, can have different responses depending on their maturity and individual receptor expression. Ensure consistent differentiation of all cells (e.g. by optimising differentiation time or using specific markers to assess maturity) to reduce variability in response.
5. You also mentioned that problems with saline and DPBS cause cell stress, but the differentiation medium seems to maintain cell viability better. The composition of the imaging medium, such as ion concentrations (e.g. calcium and magnesium) and pH buffering capacity, can significantly affect cell response. The differentiation medium is likely to provide growth factors or supplements that help keep the cells healthy during imaging. You may wish to try a modified imaging buffer that mimics the composition of your differentiation medium, but is more suitable for fluorescence imaging. FluoroBrite DMEM or BrainPhys Imaging Buffer could be good options as they provide the necessary nutrients with minimal background fluorescence. Alternatively, you could use HBSS with supplements such as glucose and calcium to better mimic the conditions of the differentiation medium.
6. Finally, if you notice a general increase in calcium levels without oscillations, the cells may be under some kind of stress (possibly metabolic or mechanical). Ensuring gentle handling, using a low-power objective (20x is reasonable, but reducing the laser intensity might help), and minimising time out of the incubator might improve consistency.
I hope these suggestions help to optimise your calcium imaging experiments.
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Good morning, working on patient serums, I noticed that in western blotting tests the IGs give false positives and unspecific signals. Ask if anyone can give me a methodology to remove these immunoglobulins, considering I have few serum available.
Thank you.
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for small volume of samples, using protein L conjugated beads for immunopreciptation is the best way to remove Igs from the blood samples. after IP, take the top for your western blot..
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Hello, could anyone help me with the protocol of deparaffinization, rehydration, and antigen retrieval for human brain tissue?
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Very straightforward.
Make sure brain sections have been thoroughly dried, at least overnight at 40°C. If struggling with sections floating off give them a bake for half an hour at 60°C before moving to next step.
3 x 5 mins in xylene- go longer if you're still seeing waxy deposits, it won't do any harm.
3 x 5 mins in IMS
5 mins 90% IMS
5 mins 80% IMS
5 mins 70% IMS
water
We tend to use citrate buffer (pH 6) as a starting point for AR. Preheat buffer in closed container in a steamer (or whatever heating method is available to you as long as the buffer isn't allowed to boil) then slides in and heated for 25 mins then allowed to cool down slowly.
This is a very successful method for both IHC and IF in our lab. Happy to answer further questions.
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Hi everyone,
I have been having some issues with SHSY5Y cells that I never had in the past. I used to culture them in DMEM:F12 with 10% FBS and 1%Antibiotic-Antimycotic and they grew just fine. Back in October, when we received a new batch of medium, I started seeing increased cell death after 1-2 passages: specifically, I would see debris like structures and the cells literally peeling off from the bottom of the flask. I initially attributed this to the quality of the medium and ruled out incubator, FBS etc issues. I thawed a new vial of cells and I had the same issue. Then I was told to try OptiMEM and with a new vial from ATCC I noticed that they grew beautifully and reached confluence within 3 days. After the 3rd passage, I started seeing the same type of pattern that I saw before--cells fragmenting, a lot of debris and cells peeling off and floating in suspension 1-2 days after plating. I am desperate as my lab has wasted many cell stocks and money on trying to figure out what this is. My next step is to test for mycoplasma but I wasn't sure if this is the case considering that we literally just bought a new stock from ATCC and it grew fine until the third passage.The medium doesn't yellow out and it is not yeast contamination. What can be the source of this type of behavior?Any input is greatly appreciated! :)
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Hi! I'm currently facing the same issues. A colleague from another lab suggested we try ultra low attachment flasks with valves. Hope this helps.
Best regards!
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We are going to overexpress neurotrophic factors in the striatum and substantia nigra region of mice. We are interested to have overexpression in both neurons and glia. The viruses are intended to be administered sterotactically.
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Several AAVs are known to affect presynaptic release regardless of what their cargo is (namely AAV1, 5, 8 see Jackman et al., doi: 10.1523/JNEUROSCI.4694-13.2014). AAV9 was the exception and transduces neurons well so after that publication many completely stopped using 1, 5 and 8. PhP.eB and PhP.AX are newer variants that cross the blood brain barrier to transduce most of the rodent brain after i.v. administration. So if you want this route as an option choose one of them - just they don't work in every mouse strain! It is always a caveat that there could be 'side effects' of transduction. For PhP.eB and PhP.AX we haven't seen any but that might be due to a failure to look in the right place! We haven't done a Jackman-type study of release for instance. Also quality of AAV preps can vary - if you see a lot of microglial activation it might be the quality and not due to the AAV per se...
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On the RG, I came across two very rarely talked but very important topics.
Abstract TP165: Associations Between Parkinson's Disease and Stroke by @Benjamin Kummer et al 2017 and Risk of stroke in patients with idiopathic Parkinson's Disease by @Claudia Becker et al 2009.
There is not much research available on these hidden topics...
Let me open the stage to unfold scientific research ideas around the world.
Topic of discussion - Does stroke or PD, have a higher chance of causing another disease!! Pharmacological models are available for their research.
For reference -
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"The strokes associated with Parkinsonism are termed small vessel strokes as they aren't normally catastrophic. 1 It typically takes several small strokes to produce the symptoms of vascular Parkinsonism. 2 Diagnosis of small vessel strokes can be confirmed with tests such as CT or MRI of the brain."
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The importance of disease classification studies for the development of diagnosis & prognosis systems is crucial. Here the main problem is the lack of available medical datasets to use.
For EMG diagnosis, many studies used EMGLab datasets, but the website is NOT available currently.
And I can't find any open-access EMG diagnosis dataset. Parkinson's, SMA, ALS, or other. There are only hand gesture detection datasets present.
Other options are
1- Using an EMG simulation program like EMG-GAN (I don't know how efficient and representative it is)
2- Long-term clinical study with a budget
3- Somehow finding a -retrospective- signal record from a hospital (I don't know how to reach them)
Additionally, I want to know the usability of this short-term prototyping approach :
Simulation of signals by mimicking the patient movements, captured by sEMG device purchased. Can this, highly questionable and insufficient method, be used for the first analysis?
I want to hear your suggestions, thoughts, and shares about the topic :)
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Qamar Ul Islam Salam Alaikum again, thank you for your kind answers and suggestions, and I apologize for my late return.
1) I don't see any pathology information in the NIH-EMG article, what was the name of the project?
2) I can't find that multi-class pathological EMG data and related article from Peng in JBHI journal, can you share the article name? When I looked at JBHI I found ECG study but it is not what I am looking for. And of course there were various EMG gesture articles.
3) I can not see any pathological EMG dataset in PhysioNet, there are only 3 signal examples from myopathy-health-neuropathy.
4) What you suggest about partnering a local hospital , how to reach them and retrospective data sharing ?
Also I reached a dataset from a Github repository but the file format is .BIN and the required programs are a bit problematic. Here I am sharing the issue link:
I found the WFDB very hard, and I am still looking for available EMG datasets.
Waiting for your answers.
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Parkinson Disease (PD) is a degenerative disease that affects motor function and sequential.
At which level of H&Y stages will this improve?
Short term or long term effect?
Any evidence to help patient and client?
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Dear Amirul AB , this article may be of interest to you.
Spinal implant helps man with advanced Parkinson’s to walk without falling
“I would fall five to six times per day,” says architect and former mayor Marc Gauthier, reflecting on his life before receiving a highly experimental implant that delivers electrical stimulation to his spinal cord. Gauthier has advanced Parkinson’s disease, and the technology enables him to walk fluidly — something no other therapy can do. Researchers say larger studies are needed to assess whether the device will work for others with the disease...
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I have UNM dataset for Parkinson's disease which is publicly available in .mat format. I'm trying to work on it using MNE python and for that I need to get the spatial distribution of the electrodes to generate MNE object.
so far after using scipi.io to read the data I've got following format for each electrode
['FC5'] [] [[-69.332]] [[0.40823333]] [[28.76282344]] [[76.24736451]] [[24.16690699]] [[69.332]] [[16.518]] [[85]] [[6]] []
about last two entries 85 is same in all 63 electrodes and 6 here is electrode index but what all these other numbers supposed to mean? where are the coordinates? Can someone explain?
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Based on the information you provided, it seems that you have obtained electrode information for each electrode in the UNM dataset for Parkinson's disease. However, the information provided does not seem to include the spatial coordinates of the electrodes.
The other numbers in the electrode information may represent various electrode properties or measurements, but without additional information or context, it is difficult to say for certain what they represent. For example, the first number in the third set of brackets may represent the electrode impedance or resistance, while the numbers in the other brackets may represent other electrical properties or measurements.
To obtain the spatial coordinates of the electrodes, you may need to consult additional documentation or metadata associated with the UNM dataset. This metadata may include information such as the electrode locations or the electrode montage used to acquire the data.
Alternatively, you may be able to infer the electrode locations from the electrode labels themselves. The electrode labels typically follow a standard naming convention, such as the 10-20 system, which specifies the approximate location of each electrode on the scalp. By using this information, you may be able to estimate the spatial coordinates of the electrodes.
Once you have obtained the spatial coordinates of the electrodes, you can use them to generate an MNE object in Python. The MNE Python library provides several functions for reading and importing electrode coordinates, such as mne.channels.read_custom_montage() and mne.channels.make_standard_montage(). By using these functions, you can create an MNE object that includes the electrode locations and can be used for further analysis.
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I am working on a project that requires the measurement of neuromelanin purity, and I have found papers that say that they have measured the purity, but they do not say how ( ).
Neuromelanin does not have a commercially available 100% standard, so I cannot do typical comparisons. Does anyone know where to begin or how to do the purity measurements?
Any help would be appreciated.
Thank you!
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There are several methods for measuring the purity of neuromelanin, including:
Spectrophotometry: This technique can be used to measure the light absorption of neuromelanin at different wavelengths. The purity of neuromelanin can be determined by comparing the resulting spectra with reference spectra.
Chromatography: This technique allows the separation of different components of a brain tissue sample using appropriate solvents. The purity of neuromelanin can be determined by analyzing the chromatographic peaks corresponding to neuromelanin and comparing their intensity with other components.
Electron microscopy: With this technique it is possible to observe the structure of neuromelanin at a microscopic level. The purity of neuromelanin can be determined by analyzing the morphology and size of neuromelanin granules.
It should be noted that measuring the purity of neuromelanin can be difficult because there are other similar pigments in the brain, such as melanin and lipofuscin. Therefore, it is important to validate the obtained results using different methods and compare the results with known reference samples.
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Dear community,
For my master's project, I want to design an experiment that will asses the efficiency of an antioxidant to stop/restore mitochondrial oxidative stress/redox balance in a PD mouse model, in both early and later stages of the disease. Redox balance will be assessed by using a roGFP lifetime measurements in vivo.
I was thinking to use MPTP model, due to it's effect on the mitochondria, simplicity and practicality. However I heard that this model can be difficult and dangerous to work with and might kill the mice faster than the experiment will end.
There is 6-ohda model, but I was reticent to use it because it has to be injected into the brain directly (which I believe might interfere with my fluorescence measurements) and the mechanisms behind it are not fully understood.
I would highly appreciate if anyone could share their experiences about working with these(or maybe other) models and give me an appreciation of their suitability in regard to my research question.
Thank you!
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* 6-OHDA and MPTP model are the most widely used animal models for screening of drugs against Parkinson's disease.
* 6-OHDA model requires stereotaxic surgery expertise, much more complex and mortality is high. Produces free radicals that acts as potent inhibitor of the mitochondrial respiratory chain complexes I and IV. Neuroinflammation and neuronal death may progress to other regions of the brain if surgery is not perfectly.
* Whereas MPTP model is simple, 25mg/kg in C57BL/6 mice for 5 days intraperitoneal. MPTP is metabolized into the toxic cation 1-methyl-4-phenylpyridinium (MPP+) by the enzyme monoamine oxidase B (MAO-B) of glial cells, specifically astrocytes and accumulates within SNpc DA neurons, where it inhibits ATP production and stimulates superoxide radical formation.
So, you can study mitochondrial dynamics, dysfunction and redox changes according to your study design.
Animal mortality would be around 10-15%. Yes MPTP is quite dangerous to work with, however I have been using the same from the past 6 years, I have attached the paper that discusses in detail, precautions needed to work with MPTP. If you can these necessary precautions, there is nothing to worry about more then.
* I would recommend you the MPTP induced PD model for your work.
Hope it works.
Thanks
Samir Ranjan
Ref:
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Hi everyone,
I am currently trying to screen the behavioral effects of the 6-OHDA unilateral injection in the striatum of Wistar male rats to confirm a Parkinson's animal model by apomorphine-induced turning behavior test (or rotameter test).
Although the dose of apomorphine is prepared according to the articles and is injected intraperitoneally to the rats, but instead of turning against the injection site of the neurotoxin, the rats become extremely relaxed and even fall asleep, and it is not possible for me to check the behavior.
I have request from dear students, professors and scientists that help me to find out what the main problem is and share the tips of rotameter test with me.
Please feel free to have contact with me.
Thank you for reading and helping!
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Please find the detailed protocol attached.
Thanks
Samir
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Does anyone know what is the first paper published about physical therapy for Parkinson's disease in the world? If so, can share the reference (or the pdf file)?
I appreciate if you could help me!
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Thank you Thomas Gloor-Juzi !
Follows below the reference I found. However, I am not sure this is the oldest reference.
CHRYSTAL M, HOFFNUNG A, BARSKY M, ROY JB. Possibilities and limitations of rehabilitation procedures for paralysis agitans. Phys Ther Rev (1948). 1952 May;32(5):231-5. doi: 10.1093/ptj/32.5.231. PMID: 14920202.
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Hello everyone,
I am using retinoic acid to differentiate SH-SY5Y cells into neuronal cells to generate a Parkinson's disease model. I will perform MTT assay before using a neurotoxin of interest to generate a PD model again for another set of experiments but I am not sure if I have to differentiate the cells again because I have seen that the same cell line is used undifferentiated as well, for example in Alzheimer's disease research.
Is it a "must" for in vitro PD model?
Thank you
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Dear Dr Samir Ranjan Panda Thank you for the information.
I performed neurite growth analysis and TH staining in differentiated SHSY5Y before and obtained results with no problem. Considering the info you gave, it seems ideal to me to differentiate the cells for the MTT protocol as well.
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Dear Researchers,
What is the best way to deal with imbalanced Dataset when the Imbalanced ratio , IR is more than 4.5 (majority 2000, minority 400) ? If I want to use deep learning methods or transfer learning for classification, what is the best way to augment the data. In my problem Healthy control dataset is the minority.
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Mosarrat Rumman You can use generative models to generate new examples for classes with less samples. Adversarial learning, and denouncing auto-encoders are powerful tools for learning robust meaningful features.
Here is an example of our designed denoising auto-encoder:
Also you can find some codes here to start with:
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Hello everyone! I am curious as to whether we can induce mitophagy in HEK293T (or HEK293, which should be similar?) cells, without Parkin transfection?
I have read some papers in the mitophagy field and got an impression that many of them use HeLa cells, which don't express Parkin endogenously, as the model cell line to study mitophagy. For mitophagy induction, they needed to transfect Parkin into the cells and exert chemical treatments afterward. And I was confused why they use HeLa cells to begin with.
If anyone has had success in studying Parkin/Pink1 mitophagy in HEK293T cells, could I get some advice as to what chemicals you use and the duration of the treatment to induce mitophagy? Thanks very much!
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I am also curious about your work. I am trying SH-SY5Y mitophagy via AAV transduction and siRNA-induced knockdown of Parkin?
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I start learning about synchronization between different states and criticality in Parkinson's disease. Would it be possible for someone to recommend a relevant and practical review paper that examines the new challenges arising in this field?
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Dear Sheida,
Here, we discussed the role of cortico-subcortical circuits in the emergence of abnormal beta oscillations as well as abnormal synchronization in PD:
The relation between patterned neural activity and abnormal synchronization in PD is briefly discussed here:
A reference review article can be found here:
Also, take a look at:
Good luck,
Best regards,
Mojtaba
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I am working on EEG self-detection of Parkinson's disease and I would need to apply my methods on data from people with Parkinson's disease subjected to ERPs or ssEVPs with a control group of healthy people subjected to the same stimulation under the same conditions.
I have been searching for a year without being able to find a database. So I am looking for a direct link to a laboratory or researcher who can provide me with this data.
Thank you.
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Hi,
See this site, it may help you:
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.
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I'm studying with a-syn preformed fibril for obtaining Parkinson's Disease model in rodents. We are trying to probe-sonicate the fibril at room temperature using different settings to obtain a fibril fragment within 40-50 nm. So, we tried to adapt your protocol (Generation of Alpha-synuclein Preformed Fibrils from Monomers and Use In Vivo) for our sonication trials; however, we couldn't reach the desired smaller diameter.
Details of our trials:
Trial 1: We used 20-25% amplitude (power of our sonicator 70W and diameter of probe is 3 mm), total 30 sec (1 sec on, 1 sec off) for 5ug/ul PFF solution, but according to the zetasizer result, we obtained fibril fragments of 505 nm average size. In the second step, we extended the time, making it 2 minute. But again, the fibril fragments were average 930 nm. Finally, we diluted the solution to 2 ug/ul from 5 ug/ul, we tried %20-25 amplitude (power of our sonicator 70W), total 60 sec (1 sec on, 1 sec off). Zetasizer results were 470 nm average size.
We thought our sonicator is weaker than in the protocol, so we tried to use more powerful a sonicator than previous at trial 2.
Trial 2: We used %30 amplitude (power of our sonicator 500W and diameter of probe is 2 mm), total 60 sec (1 sec on, 1 sec off) in 5 ug/ul and 2 ug/ul PFF solution. Zetasizer results were 549 nm average size. After then, we continued to sonicate for 2 more minutes (the total 3 minute – 1 sec on 1 sec off). Measurement results were 918 nm as average.
If possible, could you provide us with any suggestions to sonicate the fibrils?
Thanks for your helps.
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Hi, i work with fibrils in rats. Our labs and others have found that probe tip sonication is less efficient than water bath sonication at obtaining smaller fragments. With probe tip sonicators you can sonicate only for so long before you run the risk of over heating the sample. For more detail please seek out published protocols and also a best practices paper by Laura Volpicelli-Daley.
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8 µg (in 1.6 µL) of 6-OHDA was introduced into the right striatum of mice according to the coordinates A = 0.5, L = 2.0, and V = -3.5 relative to bregma. The needle was withdrawn 5 min after the injection. Seven days after operation, 0.5 mg / kg apomorphine was used to induce rotation, Unfortunately, only a few mice were able to induce seven rotations per minute. Can someone please tell me what went wrong? How can I improve the surgical or experimental protocol?
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Did you verify surgical location? You should verify canula placement prior to behavioral testing. Then again after the experiment, you need to verify placement and the effectiveness of the lesion. Stain for a marker of dopaminergic phenotype.
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We have published today a paper at Springer Nature journal of Scientific Reports entitled "Optimal time lags from causal prediction model help stratify and forecast nervous system pathology" by Theodoros Bermperidis and co-authors from my lab at Rutgers, colleagues at Stevens Institute of Technology and Columbia University. The paper describes how to standardize signals from wearables and make causal predictions forecasting disorders of genetic origins and unknown etiology. These included Fragile X and SHANK3 deletion syndromes, both linked to Autism, and Parkinson's disease and Vestibular Dizziness Handicap. I wonder if we could use gait more often in the clinical settings now that we have off-the-shelf wearables at our disposal. Research shows that it would improve classification and help stratify heterogeneous disorders like Autism and Parkinson's disease. Read more here https://www.nature.com/articles/s41598-021-00156-2
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Excellent, Elizabeth. Congratulations on this groundbreaking contribution.
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I see that many papers include analyzing calcium in their methods when the differentiation of iPSCs to neurons is taking place. Why is this necessary?
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Basal calcium is usually low in the cytoplasm of unstimulated cells, most of it is stored in the ER and in the extracellular environment. A good way to assess the differentiation of iPSCs to neurons is by testing their ability to respond to external stimuli or to fire spontaneously after synapse formation in culture. These activities would determine calcium transients that can be measured and quantified - e.g. comparing the response to the baseline (stimulus vs absence of stimulus). You can check this paper about an iPSCs-derived disease model.
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Conversely, rotenone, which is an insecticide, has.
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in fly model MPTP can be used to induce parkinsons disease refer the following article for further clarification.
"Resveratrol prolongs lifespan and improves 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-induced oxidative damage and behavioural deficits in Drosophila melanogaster"
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Adverse health effects from Rotenone have been well documented in the time since the NOSB reviewed botanicals in 1994. But still Rotenone is continue to be used for organic production. Although no longer offered for sale in the USA, rotenone is still in use as a pesticide on organic farms. Among the compelling reasons to prohibit rotenone, the most heavy is it's linked to eye problems and Parkinson's disease. Astornishelly enough is to note that Rotenone's synergist, piperonyl butoxide, is prohibited from use in organic agriculture.
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Dr. Tsyrlov, I found your question interesting. I found that rotenone is associated with the induction of parkinsonism in rat models. Also, ''Rotenone will be added as a nonsynthetic substances prohibited for use in organic crop production.'' https://www.jdsupra.com/legalnews/usda-amends-the-national-list-of-54211/
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How to make a Parkinson's disease (PD) model of Zebrafish ?
Is there any detail protocol ?
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Hi Achmad,
There are also MPTP models. Simply google "MPTP Zebrafish" and you'll find several papers using the drug model.
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I am trying to figure out how to differentiate between the pars compacta and pars reticulata. In healthy patients you can easily see the difference between the both where pars compacta has a lot of dopaminergic cells and pars reticulata doesn't. However when I look at the SN of Parkinson's patients, the clear/obvious/visible border isn't there anymore. The cells in the Substantia Nigra pars compacta (especially the ventral group, so closest to the pars reticulata) are degenerated. Thus, the clear border is gone. I am not sure where the pars compacta stops now and where the pars reticulata starts.
Are there more hallmarks to define the border between the pars compacta and reticulata?
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Hi Lydian,
We have used morphometry to distinguish between pars reticula and compacta, verified by regional analysis using a steriotaxic atlas, in rodents! Whilst we have done some of this work in non-human primates, similar work in humans brain is sadly, very limited. The key may be to use, if you guys are set up for it, multiple labeling immunofluorescence (as suggested by Max), possibly backed up by morphometric analysis. I have added a manuscript which may be useful. Wish you the best!
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Is the motor complications in Parkinson's disease are due to early use of L-Dopa or due to progression of the disease
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Extended use of levodopa can result in the patient experiencing dyskinesia (involuntary movements) and motor fluctuations (where the patient experiences “off-time” periods, as the drug, wears off and symptoms re-emerge).
Also, Parkinson diseases are progressive diseases
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Hello! I'm a Physical Therapy student and currently working on research about stabilizing spoons and their effects on people with PD and ET. I was hoping that some of you may have known any systematic review or RCT articles that I could use as a reference for my study?
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Thank you, Aedrian Abrilla! I did some research beforehand, however, I was trying to look for RCT or systematic review studies but I can't ask for a full-text request of the article. Thank you again! The links you put surely helped.
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Purely asking out of curiosity - we have another protocol that seems successful, so I'm just curious chemically speaking why this might've happened with our first protocol. We initially used a mobile phase of pH 3.0 (MDTM Type II Mobile Phase), and a 2-channel analytical cell set to -100mV and +250mV, where peaks are quantified from the +250mV electrode chromatograph. Despite being the same concentration, HVA peaks are always CONSIDERABLY shorter than all of the other monoamines and metabolites, and consequently detection cuts off at a concentration about 10-fold greater than the other neurotransmitters.
Given the pKa of homovanillic acid (4.4), compared to NE(9.5), DOPAC(3.6), DA(8.93), 5-HIAA(4.22), and 5-HT(9.31), they should all be fully protonated at a pH of 3.0 when suspended in our mobile phase, so I'm not sure why HVA shows such unique differences from the others? Our recording electrode is set to a positive cell potential, so it should be oxidizing (taking electrons from) the sample - could it be that HVA has less 'available' electrons? If so, why is that?
We have supplies coming to switch to a different mobile phase, install a conditioning cell and tackle this problem, but I'm curious to hear why mechanistically this may be happening? Also, again out of curiosity, does anyone know what about a conditioning cell (installed between the column and analytical cell) makes it capable of increasing sensitivity?
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Hi Michael, did this answer your question? Regards, Hendrik
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I am doing research for Parkinson's disease gait analysis. Where can I find a database based on the acceleration of gait from Parkinson's disease patients?
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Patient with neurological problems have different problems. This will impaired their physical, cognitive and even social interaction and so forth.
So, which approach and why that approach is used?
Look into more discussion and information.
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Task-based interventions are highly effective for gait and ambulation among patients with chronic CVA and TBI provided that a sufficient intensity is maintained.
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I am currently working with SY5Y cell line and my goal is to differentiate them into dopaminergic-like neurons for investigations related to Parkinson`s disease. As one of the markers of dopaminergic neurons I have considered using D1 receptor, but I haven`t been able to identify a single study that expressed this type of dopamine receptors in SY5Y cell line by chemical treatment (e.g. retionic acid or TPA) Many did prove expression of D2 and D3 receptors. So I am wondering should I use D1 receptor at all as a marker of dopaminergic neurons?
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Hi Ivan:
People have reported that DRD2 receptor presents in SH-SY5Y cell line, which is upregulated in retinoic acid differentiated SH-SY5Y neuroblastoma. However, for DRD1 receptor, it could probably be expressed also in SH-SY5Y according to the experiment about extracellular dopamine induces the oxidative toxicity of SH-SY5Y Cells by Jian et al. in 2008. However, basically, most of studies use exogenouse expression of DRD1 receptor for the the internalization or characterization of DRD1 receptor in SH-SY5Y cell line, which indicates lower or no expression of DRD1 receptor in SH-SY5Y cell line.
For your reference
Hope this could helps you.
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We would like to roughtly assess the potential neuroprotective ability of a certain agent to prevent people from developing Parkinson's disease (PD). PD has an incidence of 1% in the general population over 60 years. We are planning to conduct a retrospective study on a group of people, who have all been exposed to this agent for many years. The potential beneficial effect of the agent would also consider information about time of exposure to the agent and the estimated total received dosage of the agent during the lifespan. In case if the agent has neuroprotective properties, we predict people who were exposed to it for a longer period at higher dosages have lower overall prevalence of PD than the general population of the same age group. Our concern is - what would be a minimum number of participants who are 60 years old or older, to be able to estimate the agent's potential beneficial effect?
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Hey there,
You need to run a sample size estimation based on your study design. You also need to define/know the alpha (commonly 0.05) and beta (commonly 0.2) levels as well as d. Moreover, if you are planning to run a between-group analysis (case/control as I suppose), you need to know the prevalence of the feature in both your groups separately. I recommend using G*Power (free) or PASS (needs payment) software for a smooth experience. Bests
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If I'm right, dopaminergic neurons in substantia nigra but not in ventral tegmental area are affected by Parkinson's disease. I have stained brain slices with TH to observe changes in dopaminergic neurons in Parkinson's mouse model. In the confocal images obtained after staining, I could not demarcate dopaminergic neurons belonging to the substantia nigra and ventral tegmental area. I'll appreciate it if you could suggest to me the way to find the TH-labeled neurons affected in Parkinson's diseases. I have attached images I obtained and I use ImageJ for TH positive cell counting.
Thank you
Saroj
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Hello,
according to a paper from Thompson et al. in mice (!) you can perform double stainings for TH and Girk2 to determine dopaminergic neurons from the Snpc from VTA (Thompson L, Barraud P et al. (2005) J Neurosci, 25: 6567-6477).
I do not know if this works in rats.
Otherwise you can use the Atlas "Rat brain in sterotaxic coordinates" from Paxinos (or the Mouse Atlas for mice). This can help you to delineate the Snpc from VTA.
Kind regrads
Stefan
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Environmental toxicants induces PD in animal models, one of which is rotenone. Controversies exist on whether the stereotaxic technique or systemic administration is the best. While some have argued that there is high mortality using the systemic method compared to the stereotaxic surgery method. Which method best replicate the pathological hallmarks of PD?
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Thanks
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I'm a retired research chemist/physicist with Parkinson's disease and I've been working on upregulating Nrf2 with sulforaphane to fight oxidative stress.
The results I achieved on my PD and with a group of 8 people with PD have shown that sulforaphane strongly attenuated non-motor symptoms especially fatigue and lack of motivation, suggesting that the first target for Nrf2 seems to be mitochondria. I am not an expert in this subject and I would like to reach out to research groups and Parkinson's disease specialists to discuss how to take this idea further.
You can find the background here :
And a preprint I have written on RG here:
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I have been using 400 micrograms twice a day. Brand: Swanson.
I have had the drug checked at the University and they confirmed it is 87% pure sulforaphane. Not the 99% the lab says. But is good enough for me.
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Hi,
I'm working on poison induced Parkinson in rats and I do apply some behavioral tests to check Neuropathic pain.
As a result, I find out that they simply show signs of neuropathic pain ( increased mechanical allodynia meaningfully) exactly on the same side I applied lesion to VTA, Substantia nigra and striatum through stereotaxic surgery.
Knowing that there is decussation (crossings in the form of an X, relate each side of the brain to the opposite side of the body.) As a physiological normal situation.
Does anyone have the same experience?
Or Does anyone can explain this complicated result?
I assure you that I have done surgeries and behavioral tests with certitude.
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Martha I. Dávila-García Thank you dear Martha, very well explained. Firstly, I thought SNpc unilateral lesions will cause nonmotor symptoms such as pain on contralateral side of the body. So I performed experiments 3 times and I obtained the same results each and every time. Also I found out with disease progression neuropathic pain was observed on the both sides.
Again thanks for your answer. I appreciate it.
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I am trying to assess spatial memory in MPTP induced Parkinson's disease C57Bl6 mice using a Morris water maze. I wanted to know if there are any specific dimensions to the MWM to be used. Most papers suggest using 120 cm diameter with 40 cm height . Has anyone used a maze smaller in diameter than this?
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@ Ghulam Md Ashraf - thank you for your response.
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I have tried administering rotenone intraperitoneally to C57Bl/6 mice in my lab, but there is high mortality rate. Subcutaneous rotenone at a dose of 2.5 mg/kg for 28 days did not show any changes in alpha synuclein levels. I m in need of an acute rotenone model of PD, in which I can check the effect of molecule against alpha synuclein aggregation.
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choosing sc. injection was a good decision. However, 28 days is not enough. extend the days to 40-50 days and you will probably see the Lewy bodies. You can visit my latest article in this regard:
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I have some files about PET-DAT in patients, having parkinson disease. And I use spm12 implemented in matlab15b for coregistration and spatial normalization of images and voxel-based comparison. But I do not know how to measure regional uptake values for region of interests, like putamen, caudate.
Can you offer some methods and Suggestions?Thank you very much for your help.
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You nees ROIs of the regions.
Marsbar will do the trick
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Hello Research Gate community,
we are currently trying to differentiate SH-SY5Y cells into dopaminergic neurons. However, every protocol we tried so far did not work out. We tried to induce directed differentiation using RA for 5 days following supplementation with TPA for 5 days in various concentrations (as recommended in several papers). We tried differentiating using RA for 5 days followed by 5 days of BDNF treatment, which also did not work out. Are there more things we have to keep in mind as for example low glucose media or supplementation with Glutamine? We usually use standard DMEM with FCS and Pen/Strep. We would be very happy for any advice.
Thanks a lot for your suggestions.
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Firstly, you need to plate your cells as per your experiments, be careful about the density of the cells you need to differentiate. If you plate much cells they won't grow neurites or processes and they will die if you plate too less!! You should plate cells so that they can communicate! Secondly, for your ref, plate 100,000 cells in one well of 6 well dish in complete medium with 10%FBS!! Next day, add RA in Medium with less serum (1-5%) and it works really well. Change the medium every alternate day and be careful about RA, because retinoic acid is very sensitive to light exposure and after 5 days add BDNF along with RA in medium with no serum!! Thirdly, we didn’t see TH levels, but other papers have shown good images of TH in Blots and Icc
hope it works for you
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Current genomics tools to to discover new or study current biomarkers of neurodegenerative diseases.
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Do you know what is metagenomics? And did you read the question well?
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Hi Jung-Min Lee, sorry to bother you, but we are developing a project to study Physical Activity in Parkinson Disease patients, but we can not download the specific software to work with the sensors. So, can you ask the person who worked for the Sensewear Armband how is it possible to download the software needed to work with the system (sensors)?
If you intend to put me in contact with that person my email is
Thank you.
Best regards, Filipe Melo.
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Filipe M. S. Melo There is a typo on the website, try intalling the 7.0 file which actually contains v8.1 named sensewear-8.1.0.22-20140915.exe
Nara da Silva Dias I see that the main issue is that the hyperlink to install MS visual 2008 is no longer a valid url. I found a workaround and got it to work eventually by following these steps:
1. Go to https://www.microsoft.com/en-gb/download/details.aspx?id=29 and install the "vcredist_x86.exe file by clicking the download button and selecting that file.
2. Install vcredist_x86.exe which is the visual package required for Sensewear.
3. Go to "sensewear-8.1.0.22-20140915.exe", right-click and go to properties > compatability tab and change the compatability mode to Windows XP(Service pack 3), click apply.
4. Try running the installation file again.
Please let me know this works for you and please recommend this answer so that others can see it.
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Patient diagnosed with intractable major depression. Olanzapine treatment caused severe drug-induced Parkinsonian symptoms. Olanzapine discontinued 18 months ago and patient recovered from DIP. What alternative medication would you recommend?
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one could take advantage from tricyclic antidepressant: used cautiously, these old but still valuable drugs might be especially useful in case of Parkinson(ism)
regards, MC
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We investigate the mechanisms of Parkinson's disease and do drug design for Parkinson's disease. We use theoretical physical chemistry, biophysics and bioinformatics in our studies. We are in need of experimental collaborators to apply for funding from the Michael J Fox foundationand COST EU projects. Are you interested? 
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Good answer dear Orkid Coskuner
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One of the current Models prepared to cure Parkinson's disease is based on the process to derive Brain cells from Skin Cells. Is the same possible?
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Following.
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Does anyone have a tip/hint on how to deliver physical assessment remotely? References are welcome! I am working on a project to deliver physical exercises remotely to older people and people with Parkinson's disease, however the research team would like to assess participants' progress too. #COVIDー19
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Hello Paulo Henrique Silva Pelicioni,
Comfortable gait speed is a non-invasive indicator of physical condition. Aging or illness reduces the energy capacity of the body. Part of this power is spent on movement. It is equal to the force spent on movement (F) multiplied by the speed of movement (V), i.e. FV. F is roughly the same (we consider the body weight the same). Then an increase in the comfortable gait speed will indicate an improvement in physical condition, and a decrease in the gait speed will indicate its deterioration.
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Sleep problems can be seen in patients with Parkinson's disease. Can it be about serotonin?
In mice, ablation of the raphe and no production serotonin increases wakefulness and impairs the homeostatic response to sleep deprivation.(DOI:https://doi.org/10.1016/j.neuron.2019.05.038)
Even in the absence of depression, the CSF levels of 5-I-HAA of patients with Parkinson’s disease are lower than those of age-matched controls.
( DOI: 10.1176/jnp.2.1.88 )
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Thank you for great articles. They gave me a new perspective about PD.
I want to suggest an article: Serotonergic dysregulation is linked to sleep problems in Parkinson's disease (doi:10.1016/j.nicl.2018.03.001)
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since apomorphine is not availble, and pramipexole and apomorphine are both dopamine receptor agonists, so I'm wondering if I can use other dopamine receptor agonists to verify the rotation test of 6-OHDA induced PD mouse model.
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/LABORATORY ANIMALS: Acute Exposure/ A 0.1% injectable solution of pramipexole injected paravenously into the jugular vein was conditionally tolerated by rats. Single intravenous injections of pramipexole 0.1% solution into the marginal vein of the ear were tolerated by rabbits. Single intraarterial injections of pramipexole into the central artery of the ear were tolerated by rabbits. Health Canada; Product Monograph for Mirapex (Pramipexole Dihydrochloride) Tablets, Drug Identification Number (DIN): 02237145 p.41 (Date of Revision: November 27, 2012). Available from, as of June 12, 2015: http://webprod5.hc-sc.gc.ca/dpd-bdpp/start-debuter.do?lang=eng
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Why is running in Parkinson's disease both possible and have therapeutic effects in contradistinction to walking?
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I think that the attached video link might yield some interesting insights that could be parlayed into controlled investigations if anyone is interested.
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Hi Everyone. I am trying to check the neuroprotective activity of some compounds in the rotenone model of Parkinson's disease in PC-12, for which I will check the effect of that compound on cell viability first. I want to ask whether it is required to differentiate PC-12 for this purpose, as I tried to differentiate the cells using 30 ng/mL as well as 50 ng/mL NGF. I am able to see the differentiated cells in 3-4 days in 96 well plate, but NGF itself is causing cell death, which could be a variable for the cell viability test.
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Hii Suriaya. I am currently seeding 5000 cells per well in 96 well plate. I will try with 2000 cells per well for this time. Moreover, I am also getting cell death in 6 well plate, where I am seeding 5,00,000 cells per well. Please suggest if I can differentiate using less dose of NGF.
Thank you
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The association of free radicals in the pathophysiology of chronic diseases like degenerative brain disorders (AD, PD, HD, Stroke) has been evident by a substantial research.
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We are seeking to identify the major barriers Parkinson's disease patients face in obtaining care for mental health issues. What are the major gaps in treating mental health disturbances in this population?
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We had a mouse model of PD and check if exercise in a running wheel decreased the damaged neurons or stopped the degeneration of them. If you are interested I attach the paper:
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Hello,
I'm currently designing a protocol in Parkinson's disease and I was wondering about the optimal timing to test motor symptoms in relation to last dose of dopamine agonist(s).
It is clear to me that standardisation of this aspect is essential, but I could not find specific literature with recommendations in this regard.
Any pointers to papers or opinions on this matter would be immensely appreciated.
Thank you
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Thanks for your response Victor, it is very helpful and the reference to the LED is also much appreciated!
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I want to test the effect of a neuroprotective drug on rotenone rat model of Parkinson's disease but after reading the related articles about my work, I became doubts about select the best dosage of the drug and treatment methods. in some of the articles they using a low dosage of a neuroprotective drug and in some high dosage and their treatment was different in some cases, they use the oral method and some intraperitoneal injection method.
Does anyone have any suggestions to select the best dosage and treatment (o.p or i.p)?
Many thanks
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Do you know the highest dose that does not produce an adverse effect? If so, this is your NOAEL. A dose 10 fold lower than this should be you maximum dose to ensure a good margin of safety. You will then need to use this a start point for dose response analysis. Starting with the NOAEL dose you need to back down by ten fold increments to the next two lower doses. For example, if the NOAEL is 100 mg/ kg your dose response analysis should start with 1mg/kg, then 10 mg/kg then 100mg/kg. This should give you a good baseline set of data that can be translated to NHP and humans using allosteric modeling.
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Can histone deacetylase activity influence the progression of Parkinson disease? If so, what are the underlying mechanisms?
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You might be interested in our PD models:
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Current researches to cure Parkinson's disease deal with the formation of the dopaminergic neurons from the Skin fibroblasts but even after the formation of neurons, is their administration to the Hippocampus region of Brain possible?
If yes, is that economically feasible?
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Great study discussion with senior neurosciences Professor anatomy and physiology will guide you better to understand brain and spinal cord,
Skin is also very important, understand your question,
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Parkinson's Disease is a seerious issue now days.
Yoga may be a way of manage this problem. What yoga practice should be provided to them?
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The results of recent review found that practicing yoga helped improve functional mobility, balance, and lower-limb strength in people with Parkinson's disease. In addition to improved balance, flexibility, and posture, participants experienced a boost in mood and better sleep quality.
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I have found only a handful of studies in my review of literature. Just wondering if anyone is looking at this also.
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Please take a look at this useful RG link.
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According to researches, appendix might has connection with Parkinson's Disease but consequences are conflict. One study says, appendix removal reduces risk of PD but another study says that it increases.
Uncovering a Link Between the Appendix and Parkinson Disease Risk
doi:10.1001/jama.2019.9041
Thank you!
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I'm looking for the biochemical explanation behind the creation of genetically modified organisms (GMOs) for use in research of human pathologies such as Parkinson's Disease (PD) or Huntington's Disease (HD). I will really appreciate any answer or recommendation of literature.
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Remember that animals have four paws/legs on the ground but humans only two. Therefore magnetic fields from remanent magnetism or magnetism in the lab however caused will vary in bodily absorption between man and animal. EMF levels in labs can be very high - are the labs shielded ? Reactive Oxygen Species from unpaired electrons are of very high significance in adverse effects.
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I'm interested to continue with brain signal research in case parkinson disease. I'm searching for EEG data for the same or interested to work with the research groups in the same field.
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The data which I have it not on EEG - it is on gait monitoring. Sorry. If you would like I can forward its link.
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Hi, we're working on a pilot research and our aim is to develope parkinson dis. with both intranuclear and intraperitoneal injection methods.
In i.p. model The problem is rats can not tolerate the protocol recommended in articles [ examined dosages : 2-2.5 mg/kg dissolved in DMSO and diluted in MCT i.p. injection ]. We found Rats dead after 2-3 consecutive days.
What's the solution ?
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I definitely agree with trying relatively lower doses and spacing out injections like what was suggested. If this does not lower to an acceptable mortality rate, you may try another compound,
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), that works similarly to rotenone.
Have you had any autopsy done on the animals to determine the cause of mortality? It may be revealing to see if there was a direct toxic effect on intraperitoneal tissues.
Good luck!
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Need to consider all variability arising from age, gender, post mortem interval etc.
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use SPSS latest version & you will find sample size after feeding basics of your requirement into software. Than ask for sample size it will display sample size.
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Here is the latest example of our 20 years of cognitive dissonance. That the "heterogeneity of PD" remains "a major problem" but that better techniques (such as "utilization of complex analysis strategies and automated standardized assessment methods") will circumvent it. If even LRRK2-PD, as indirectly suggested by the authors, represents a collection of different diseases, can we rely on the brute force of newer and sophisticated analytic methods to bring us out of the conundrum that our unabated allegiance to PD as a unitary disease construct brings? Our field remains stuck in the quest to validate the century-old clinico-pathologic, convergent model of disease nosology, unlike most other fields of medicine, ahead by three decades of embracing systems biology divergence. No analytic technique will overcome the basic flaw in our ongoing biomarker-discovery efforts: that we, clinicians, get to set the gold standard against which to validate biology. It should be the other way around: biology validating disease subtypes. Unless we start walking the talk of PD as a syndrome of many biologically distinct diseases --we will continue banging our heads against the same wall. It is time to move beyond the search for biomarkers of convergence. Whatever is found to be common to all forms of PD is unlikely to be pathogenic in any.
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Thank you for taking the charge against this matter. I share your concerns and opine them to any willing listeners.
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I wanted to work on developing a deep Learning model to differentiate different classes of disease and controls. But I am not able to find the required database for it. Can someone suggest me where I can find one?
Or can anyone tell me how do I look for database related to particular icd_9 codes?
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We already know the application of botilinum toxine drooling in patients with Parkinson's disease. We often recommend that you swallow saliva and chew gum. What are the methods you use in your daily practice?
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You may want to consider Glycopyrrolate (Glycopyrronium bromide) as a anti-cholinergic drug. See also:
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Parkinson's disease (PD) is a neurodegenerative disorder that affects predominately dopaminergic neurons in a specific area of the brain called substantia nigra. While Parkinson’s itself is not fatal, disease complications can be serious. The Centers for Disease Control and Prevention (CDC) rated complications from PD as the 14th cause of death in the United States.
Currently, all therapies used for PD improve symptoms without slowing or halting the disease progression.
So at present what is the life expectancy of a Parkinson's patient?
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This is a very and unique occasion to partecipate at the first convention in which patients, caregivers and Doctors will work toghether in a brain storming....
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Dear Stephania,
The convention has already passed but we developed a PD model (mice) and tested the beneficial effects of physical exercise. It may interest you:
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Hi,
I've been doing several western blot trying to check protein expression, oligomer formation and protein degradation in alpha synuclein in cell lines (SH, PC12 and HEK) after transfection with plasmids (pIres).
I've had a lot of varibiality in my results and I don't know what I'm doing wrong, or why I have such variability in my blots that make them non consistent.
I don't use native gels, but I still see monomers, dimers, trimers and tetramers of alpha synuclein.
I've tested Old and fresh RIPA buffer. I've also tested membrane fixation with 0.4% PFA which improve a bit the bands.
Sometimes I get monomers and dimers for SH, and dimers, trimers, and tetramers for PC12, and sometimes only monomers in SH, and only tetramers for PC12.
I've get other weird results like no tubulin in PC12, or only GFP in the lane where is the C- with pIres emtpy vector only expressing GFP, but not in the rest of lanes with pIres expressing alpha synuclein and GFP (and I confirm that cells were green).
So, I'm desperate and I will appreciate all your help, please!
Attached the procedure and also some images.
Thanks!
The detailed protocol is:
Cells in 24 well plate as C- or transfected with pIres plasmid with alpha synuclein-6xHis tag and GFP
- Remove medium
- Trypsinize and inactivate. Collect in a eppendorf and add 500 ul of PBS to wash them
- Centrifuge 5 min 300 g RT
- Aspirate the SN and freeze the pellet in dry ice. Store the samples at -80ºC
- Lysis buffer: RIPA (50 mM Tris-HCl pH 7,5; 1% TritonX100; 1% NaDeoxycholate; 150 mM NaCl; 1 mM EDTA; 10 mM NaF) + 100x Halt Inhibitor
- Add 100 ul per sample of lysis buffer and vortex until pellet get resuspended. Incubate 30 min on ice and vortex at minute 15.
- Centrifuge 10 min at 4ºC 10.000 rpm
- Transfer the supernatant to a new epp and store the SN and the pellet at -80ºC until use.
- BCA quantification using 10 ul of sample
Western blot:
- 5-10 ug of sample + 4x loading buffer (10% b-mercaptoethanol), 95ºC 10 min + spin
- Load the samples on Precast Gel TGX Biorad (4-20%) Electrophoresis: 100 v 1h 30 min
- Open the cassete in transfer buffer and place the gel over the PVDF membrane (BIORAD already prepare for Tran Blot Turbo System (semy dry system). I usually soak the transfer cuvette, filters and membrane and remove the excess of buffer. Transfer of a mini (2.5A cte up to 25 V 3 min) or a midi or 2 gels (2.5 A cte up to 25, 7 min) gel to PVDF membrane
- 1x TBS wash
- 0,4% PFA fixation 30 min RT
- 2x TBS wash
- Ponceau staining 5 min
- 3x TBS-T wash
- Blocking 5% milk in TBS-T (1-2 h RT)
- Primary antibody rabbit anti alpha synuclein (PA5-17820) 1:1000 in TBS-T ON at 4ºC
- 3x TBS-T wash
- Secondary antibody goat anti rabbit –HRP 1:40.000 1-2 h RT
- 3x TBS-T wash
- Dry excess and put in a dry tray
- Develop with SuperSignal West Pico PLUS Chemilluminiscence (2 ml of mixed buffers and incubate 5 min).
- Remove the excess of substrate and place it between transparent plastic
- Imaging with Amersham 600RGB (1sec, 1 min and incremental ever 1 or 3 or 5 min....)
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Hi Ana, I'm having some difficulty following your experiment, a bit too many uncertain points... then of course giving you any suggestion is difficult. So let me do some troubleshooting and ask you a few questions: 1) How is your plasmid engineered? Who is controlled by the IRES, the GFP (I hope so) or the alpha-synuclein? (This could explain why you lose expression of the GFP in the full vector, if the Cap-dependent translation is working well, the IRES-dependent translation will be less efficient. But if there is no CDS before the IRES, there is no competition on the mRNA and the IRES-dependent translation will work better. IF is more sensitive than WB, for this reason you probably detect some fluorescence at the microscope. Moreover a smaller plasmid might also be better transfected into cells, so more copies could enter a cell or more cells be transfected. Lastly, they are two different plasmid preps and one might have come out cleaner, one less, affecting differently the transfection efficiency - seems the case of the GFPblot image, top blot, see point 2) 2) Which cells are expressing endogenous alpha-synuclein? I can see that SH cells do (you see the doublet at 20 kDa, the top band being your His-tagged version, in the Nooligomers picture, top blot). This means that in the top blot of the GFPblot image you did not transfect the cells, or the transfection efficiency was too low (few cells, or too little plasmid per cell), and the His-tagged band is missing from the WB. Anyway the GFP band in the C- is already pretty weak...). PC12 cells also express alpha-synuclein, but it's rare to see your His-tagged version and the endogenous in these cells, anyway in the bottom gel of the GFPblot image something went wrong with the WB or the PC12 extracts, all signals are too low, and when a-synuclein monomers are so weak you can't expect to detect GFP expressed from an IRES. Finally HEK cells... impressive, they seem to express low levels of a-synuclein, all your C- controls show a weak band at the right height. Anyway, it's a good control to verify that you are transfecting your cells. Secondly, they never show tetramers... Possibly the formation of the multimers is concentration-dependent... 3) The multimers: What you see at 75 kDa, ONLY in PC12, I personally doubt it's tetramers, it seems a non-specific band from the Ab recognizing something in PC12 (in the Notubulinin PC12 image you have this band in the C- where you don't even detect the tranfected monomer nor the endogenous...). Anyway, if you have sure evidence that it's tetramers, keep in mind you see them only in PC12. You are boiling the protein extract in SDS and you are running a denaturing gel... it's difficult to imagine aggregation of such a small disordered peptide... Also what you call dimers, which you detect at the same level in transfected and C- samples or with stochastic intensity levels.... very suspicious... Have you ever checked your Ab on a a-Syn KO or KD extract? I would do it... or test a different Ab... So, you are right, it looks quite chaotic. You also seem to have protein degradation, at least sometimes (the 25 kDa tubulin band in the bottom blot in the Nooligomers figure). To start, you can try to improve a few things in the protocol: 1) My suggestion is to lyse the cells directly on the plate on ice: remove medium, wash twice with warm PBS like if you should passage the cells, remove thoroughly the PBS and place the plate on ice. Then add the 100 ul of ice-cold lysis buffer on the cells in the well. Pipette to help the lysis a few times in the well and transfer in tube. Do not vortex, there is no need, the RIPA is strong enough (in fact the TritonX would also suffice) to lyse. You can shake the tube once in a while with your fingers or pipette while you incubate on ice. Incubate 15 min on ice. Never bring native protein extracts at room temp, always on ice. 2) Proteins are delicate! Thawing and freezing degrades them quickly, denatures them and induces aggregation depending on the protein type. My suggestion would be if you only need them for WB, immediately after lysis quantify while keeping the lysate on ice and then denature them with Laemmli and boil. Denatured proteins dstill degrade when thawing and freezing, but more uniformly and less. Store in 50 ug aliquots at -20 (so that you thaw and freeze the only 5 times). Anyway, if you wish to freeze them native, then you should shock-freeze them in liquid nitrogen. 3) WB. I had quite bad experience with semi-dry blotting. It is very uneven... and I believe you are experiencing this quite clearly. My suggestion would be to try a few blots with the submerged method. It's longer (70 min at 110 V), it uses more buffer... But you will be surprised by the result... Then don't forget what I mentioned above... test a different Ab or try to obtain or produce a-Syn knock-out or knock-down protein extracts... Good luck.
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Vedad Delic thanks for your answer
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