Neuroprotection - Science topic

Welcome

Regards

Hi.

I saw similar things in cause of bacterial contamination. You should look cells before dissolving formazan: bacterial cells are blue, very small, and often motile.

In this cause you should test all materials, witch contacted with experimental cells, and all reactives. The process of dissolving and diluting extracts you should assume unreliable.

Good luck!

Both

I have been looking into this, as well. I have seen bands at ~90kDa, at ~50kDa, and at ~20kDa. Primary literature points to the ~90kDa band as likely some SDS-insoluble oligomer. But, I can't seem to find anything for the 20kDa band. Have you found anything yet?

I will try to explain it better. The average concentration of estradiol in brain is of little value. Then, even having an excellent method of extraction of estradiol from brain tissue the data will tell you little. Unless yo are measuring pharmacokinetics/biodistribution after estradiol administration.

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

Hi Di, use the HistoGrip for SH-SY5Y differentiation is super good. https://www.thermofisher.com/order/catalog/product/008050#/008050.

HAVE A TRY.

Upto now no gold standard or best non invasive method to detect neuroprotection in humans are not available, but fMRI will help somehave.

There does seem to be some encouraging evidence of α2 agonists as neuroprotective in contexts of ischaemic insult in the CNS. See below:

I Agree With Isabel Taveira

You can check this article 👆

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

Monokesh Kumer Sen, on every sub- & microscopical illustration of CNS tissues the myelin sheaths are seen to form a sort of relatively soft buffer elements which distance the - as evident in any kind of trauma - tougher axons from the astroglial and vascular meshwork that forms the brain and spinal cord's "fibrous skeleton". Already in the 19th century the myelin sheaths had been shown to have a remarkable potential for regeneration, at least in the peripheral - and been suspected to also have in the central - nervous system ... and supposed to explain the remissions after clinical relapses of MS.

HA, I know exactly what you mean! Had similar cases and the delusory stuff is widespread thanks to the unethical "cannabiz" b.s. Would love to chat. I'm in a home office these days, based in S.Oregon ie pacific time zone. My cell is 541 326 6082. Afternoons best. Around most of the day tomorrow wed 5th but can call you to suit if you are still a busy onc nurse. Let me know what works. Best JT

Actually we have initial reduction of dose. After first 12 h 20/2+1=11, next 12 h 11/2+1=6.5, 48 h later 2.125+1=3.125 and so on. 12 days more we have stable 2 not the promised 20.

Hi Nur,

It is miscible in chloroform.

this link is useful

https://onlinelibrary.wiley.com/doi/abs/10.../j.1472-8206.2008.00580...

regards

How can I design a study to test the neuroprotective effect of a fruit using albino wistar rats?? Pls can you treat this as urgent??

Dear sir Franco.

Please elaborate a little more. My work is on rat's brain hippocampal slices. What will b the consequences?

Hi Salma Nabil,

The Rat and the Mice do not have the same LD50, so there is something to convert the dose !!

Hy,

enclosed you find an article about a study in which I used a monoclonal anti-TH from Sigma for western blotting.

Sincerely Yours

Thank you very much for your reply

Ok Dr. David Rotlant,

Thanks for your reply

This article was about increasing parkin activity, you can check it: Ubiquitination Increases Parkin Activity to Promote Autophagic α Synuclein Clearance - Plos journals.

Cuba may be a good to explore.

Hi Sagar,

Here is the protocol I use for BV2 or primary microglia cultures:

Prepare Griess reagent:

2.3ml Phosphoric acid (85%)

1g Sulfanilamide

0.1g Naphtylethylenediamine

97.7ml water

Vortex. Prepare Griess reagent at least half a day before use. Protect from light.

Keep media from cell cultures on ice.

Prepare standards from sodium nitrite (NaNO2): from 0 to 1000 µM. Use the culture media for dilutions.

Pipette media and standards 50µL/well on a 96w-plate.

Add 50µL of Griess reagent on top of each sample. Shake for 10min and protect from light.

Measure absorbance at 540nm in the reader machine.

Good luck,

Sighild

It depends on the type of damage you are goingto induce and type of neuroprotection you are trying to evaluate, but I don't think the positive control is necessary in this case. As you said it yourself, most of the groups don't use one. 

Best,

Jan

Your choice of model and toxicant depend upon the question you are asking. First define your research question or hypothesis. Then devise specific aims to test the hypothesis . Finally, choose methods to carry out the aims.

There are many indirect (behavioral) assays you could use in Drosophila. Activity, courtship activity, starvation resistance, etc. I'm sure there are also histological & biochemical assays, but that is outside my experience.

What our lab has done in the past is we would inject a high molecular weight rhodamine dextran intravenously. If the BBB is compromised you will see macrophages (iba1 antibody will work nicely) pick up the rhodamine dextran. If you don't see any rhodamine dextran on macrophages it means it is intact.

I hope this helps.

There is also an excellent new paper out from Marie Eve Tremblay showing a new type of microglia (called "dark" microglia based on their EM appearance) that becomes abundant during pathological states such as chronic stress, aging, Alzheimer's, etc. They display signs of oxidative stress, but also have highly ramified, thin processes, so it sounds like they may be relevant for your question. See paper below:

Glia. 2016 May;64(5):826-39. doi: 10.1002/glia.22966. Epub 2016 Feb 5.

Dark microglia: A new phenotype predominantly associated with pathological states.

Bisht K1, Sharma KP1, Lecours C1, Gabriela Sánchez M1, El Hajj H1, Milior G2, Olmos-Alonso A3, Gómez-Nicola D3, Luheshi G4, Vallières L1, Branchi I5, Maggi L2, Limatola C2, Butovsky O6, Tremblay MÈ1.

S1PR1 is one of the targets of the multiple sclerosis drug FTY720 (Fingolimod). Here is a review article about S1P and S1PRs: http://www.hindawi.com/journals/mi/2016/8606878/

Some more papers: 

So selective neural damage, mediated by activation of HSV1, would be triggered by an environmental toxin? Interesting idea, but in terms of translational research, you may have a long road ahead... HSV1 has been suggested to be the culprit in many neurological and psychiatric diseases, but so far the evidence is anything but convincing. On the other hand, there is no denying that HSV1 works in mysterious ways http://www.neurology.org/content/83/21/1888.short

J

Dear Sakharnova:

I found this paper "Identification of a low-molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice"

However, you can also check many other drugs here:

Best regards. Bruno

Resveratrol Stabilizes Amyloid in Alzheimer's

Pauline Anderson

| September 17, 2015  

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High doses of purified resveratrol, a polyphenol found in some foods, appear to stabilize levels of amyloid beta (Aβ) in cerebrovascular fluid (CSF) and in plasma in patients with mild to moderate Alzheimer disease (AD) and are safe and well tolerated, a new phase 2 study has shown.

Although it is too soon to start recommending resveratrol supplements to patients, the research indicates that this compound is safe and is promising, lead author R. Scott Turner, MD, PhD, professor, Neurology, and director of the Memory Disorders Program, Georgetown University Medical Center, Washington, DC, one of 21 medical centers across the United States participating in the study.

"It seems to have some interesting effects, enough to justify further research into this strategy," he told Medscape Medical News.

The study was published online September 11 as an Open Access article in Neurology.

C57bl/6 are resistant to auditory but not drug or febrile induced seizures. The morphological features of apoptosis and cleaved caspase-3 suggests C57bl/6 have quite restrictive doseresponse to seizure induction. Reference below

Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse Araki et al  8 JUL 2002   DOI: 10.1002/jnr.10356

Kainic acid (KA) effectively elicited ipsilateral CA3 pyramidal neuronal death within a narrow dose range of 0.1–0.3 μg, with mortality <10%.Focally evoked seizures in the C57BL/6 strain associated with a restricted pattern of apoptotic neurodegeneration

Indeed, a fascinating topic. Thank you Dr. Sutter to bring this up. I came across a recent article by the Cambridge/London group that has enormous experience with ICP. Intra-spinal cord pressure seems to be closely matched to epidural pressure at the level of the lesion in spine-injured patients, which would make pressure monitoring less invasive and still reliable. Regarding spinal perfusion pressure, the evidence being gathered is most convincing for a principle analogous to CCP.

I believe that further studies should be done to evaluate the mechanisms associated with decreased reactive oxygen species (ROS).

Hi Panagiotis,

I hope you resolve the problem. If not, there may be high levels of salt in your reagents.

Vinod

I would try serum deprivation.

hi, may I suggest http://www.sciencedirect.com/science/article/pii/S0163782714000289

hope you will find what you are looking for.

Hi Cheryl, 

I didn't study depression, but I know that GH-deficient untreated patients show it besides many other symptoms; I know too, my own experience as MD, that treating GH-deficient patients improves these symptoms. What is the reason for it? before trying to give an answer I will tell you that BDNF is one of the many neurotrophic factors induced by GH.

I don't think that correcting or improving symptoms such as depression, associated to GH-deficiency, might be due to the known GH effects on brain neurogenesis and plasticity, but related to the really complex regulation of pituitary GH secretion.

Pituitary GH release is mainly subject to a negative control from the hypothalamus. This is due to somatostatin; this peptide negatively controls the hypothalamic release of GHRH and its stimulatory effects on GH secretion. Therefore, somatostatin is the main controller of pituitary GH release.

In turn, somatostatin is mainly controlled by catecholamines, mainly noradrenaline that is formed from dopamine. High levels of noradrenaline inhibit somatostatin release, then GHRH can be released inducing GH secretion.

If you have GH-deficiency it is likely that somatostatin levels are decreased, consequently there is no need for increased catecholamines levels. Perhaps there is the answer to your question. 

If you administer GH, it produces a positive feed-back on somatostatin, leading to the release of this peptide. This, in turn would lead to increased catecholamines turnover....

While all of these mechanisms are real and we described it years ago in Trends on Endocrinology and Metabolism, my description about what could happen in GH-deficiency associated depression is only a hypothesis.

Best regards

Jesús Devesa

Hi Eduardo - I would definitely support Sven's comments. You can't really look at excitotoxicity properly until the neurons have developed synaptic connections and the receptor machinery of excitatory receptors has been well localised to receptor complexes. We published on this issue a few years back. You may see excitotoxic effects due to potential extrasynaptic receptors at earlier stages of development. See https://www.researchgate.net/publication/6929998_Localization_of_glutamate_receptors_in_developing_cortical_neurons_in_culture_and_relationship_to_susceptibility_to_excitotoxicity

Hi Kelly,

In our lab we are using SH-SY5Y cells for PD research. N27 rat dopaminergic cell line is another choice of ours. However, I think, one of the best options you can use is the primary midbrain cultures isolated from rat embryos. We dissect midbrain from E17 rat embryos and culture the cells. It's, obviously, a mixed culture including dopaminergic and GABAergic neurons, as well as astrocytes and microglia. You can also inhibit the growth of glial cells in cultures dishes via different treatments if you wanna eliminate these. Since it's a mixed culture, you can see the effect of your treatment/chemical/compound on different neuronal or glial cells, which might be an advantages depending on the purpose of your neuroprotective assay.

I hope this helps a bit. Good luck!

AR is highly expressed in brain tissue. I would not be surprised there if there would be a reason for that. I am happy to learn with you if there is somebody that is willing to share info with us.

The question posted is How to calculate the effective dose of a particular compound in rats?

By definition, an effective dose is the dose of the compound or agent at the target organ that has an effect at the target organ.  By your question, I presume that you do not have a means of measuring the dose of the agent or compound at the target organ that produces the effect of interest and therefore, you are looking to "calculate" the dose at the target organ.

Where appropriate pharmaco-kinetic models exist for your specific rat species, you might use them to calculate the dose at the target organ.  Such models presume that you can model the distribution and fate a compound or agent under certain conditions within the rat, but often fail to account for specific protein binding, selective or active transport, and other processes that might be specific for the compound or agent of interest.

In toxicology, scientists have devised several means of estimating the "effective dose" at the target organ from various dosing protocols (e.g., oral gavage, consumption of food or water, percutaneous injection, dermal absorption, etc.). Such estimates, while useful, often rely on assumptions that may or may not hold true for your species of rat (e.g., body weight, pharmaco-kinetic differences, etc.). Roughly, toxicologists are generally interested in the concentration of the compound or agent in blood over time, with the area under the curve (concentration v time) describing the estimated dose to the target organ.

To appropriately answer your question, I would need to know the compound or agent administered, the species of rat, the target organ and effect of interest.

Due to my experience with neurorehabilitation of patients after long-term coma with the following outcome in the apallic syndrome or in vegetative states, we observe the minimal brain electrical activity similar to serow line activity or dominant delta-wave activity with convulsive activity. We connect this brain damage not only with brain hypoxia, but also with severe ischemia, oedema and venous stasis in the brain tissue. We had a good result of the intensive medicaments treatment combine with intensive rehabilitation - the restore of the conscience, speech, memory, active moves of limbs etc. More - http://inno-health.com/istoriji-uspihu-roman-shvorak/ and http://angio-veritas.com/technologies/nejroreabilitatsiya-psyhonevrolohichnyh-hvoryh/apalichnyj-syndrom/?lang=en

Dear Colleaque,

My experience has shown that in rodents ethanol as a solvent of medicines has mainly protective effect. In particular, ethanol reduces oxidative stress of brain tissue arising from MAO-dependent oxidation of dopamine.

Like Pablo said, I think Mdzinarishvili A. et al has done a similar study quite recently. However, I believe that they give T3 30 minutes BEFORE t-MCAO rather than afterward which makes it potentially less clinically relevant. It would be nice if someone looked at it after stroke. You'll have to excuse my unfamiliarity with T3, but for the reasons you've given, it would be even better if someone delivered T3 at a later phase (during repair phase, for example) to see if it helps in the recovery process. 

I agree but would also recommend having a look at the fantastic meta-analysis package metafor for R, which includes a function to calculate effect sizes from different indicators (escalc()

Dear Lee, The clinical trial on NMDAR antagonists show failed it may be due to the randomized mixed population and moreover, I don't think the stroke can be prevented by single target. Always should go for computational therapy. As we all know there are so many drugs and reports showing protection by inhibition of different molecule through different reagent or compound in ischemia. Then how could be possible prevents ischemia by one target like NMDA receptor.

Secondly t-PA with acute ischemic stroke, why it is three-hour window because after three hours of ischemic condition goes to more damage of brain becuase it is not now the acute ischemic stroke, Its a severe and cause intra-cranial bleed and more intravenous thrombolysis.

Third, in case of sensitivity of neurons towards ischemic damage is less because glial cells initially protects the neurons in pathological condition.

As far as i know, there is no discernible difference. often in studies they will use one sex over another (in rats, typically female....just easier to handle). As Just mentioned, there is an influence of estrogens (and related hormones), but not a significant one to justify using it as a  pharmacological target.

All good over here, don't mind if I answer my own question.

We bought a Biorad electroporator and have gotten good results thus far.

Hi Gabriele,

the mentioned nanion products do not allow for the analysis of cellular networks. This product family and ALL other APC systems availbale were developed and optimized for cells in suspension, cells freshly detached from culture flasks or cells from thawn vials. These APCs cannot analyze cultured cellular networks and were not built for that issue. Let me know if that´s not right.

Don´t get me wrong, nanion and the other APC producers did a great job with their APC development. Chapeau! The devices are great if you need higher troughput. I guess the new nanion 384-channel machine will have a good response in the market. Congrats to nanion!

But if you need network data or a higher organisation grade like in a cell network (some ion channels are only expressed when the cells are attached to a substrate or to other cells...) or in a brain slice, you are limited to manual patch clamp. And we all now that using a manual rig does not allow for higher throughput (5-20 data points per day if you are very experienced). Hence, we believe (and know from our potential customers already) that our future systems are complementary to conventional APCs. Further, our system will drastically increase the parallel number of patched cells, up to 16 at once with one chip! If we get that done: world record! I am sure that the information of the cell-cell-communication analyzed with our systems will be of very high value in future.

By the way, Prof. Markram´s 1 billion € project uses a 12-channel setup (http://www.2045.com/news/31235.html). The rig can be seen in the last figure in this article. The parallel patch clamp of neurons is an important tool for his research.

Well, with our short survey we would like to know the point of view of scientists regarding our technology to define our product development when we start our company in future.

For further information, please contact me under philipp.koester@uni-rostock.de

Best regards, Philipp

Hi,

The Tocris company offers a STAT3 activator polypeptide with some interesting properties including CNS penetration and low systemic toxicity, called colivelin. Seems to be rather specific.

Good luck!

It depends upon what you want to investigate and your expertise. Several "good" models of sicatic nerve lesions have been used to study neuropathic pain, and most are useful. Particularly the spared nerve injury is probably the most consistent in terms of results. What is also very important is to use adequate methods for evaluation. I would not recommend the hot plate. Algesimetry tests for mechanical (Von Frey) and thermal (plantar) focal stimulation are adequate. However, you have to be aware of the innervation territories of the limbs and which areas are denervated or innervated in the surgical model you use. You may take a look at our recent article Cobianchi et al Exp Neurol 2014, 255:1-11.

There are multiple ways to assess neuroinflammation. You can perform immunohistochemistry by using specific markers for microglial activation (Iba1) , astrocytic response (GFAP), and neuronal loss (NeuN or Fluorojade for dying neurons). You can also look at the levels of various cytokines that are upregulated during inflammation (TNF-alpha, Interleukins, TGF-beta, and so on).

As you know LPS mediated inflammation is closely associated with nitric oxide production and up regulation of iNOS production at most sites all of which has considerable publications documenting LPS effect on inflammation. Other mediators also play a role. Systemic administration of LPS also produces change in the Nucleus Tractus Solitarii. There is a lot of literature supporting communication pathways between the immune system and brain. I have attached one for your consideration. You are treading on unknown territory as best I know and very interesting work, I look forward to hearing more.

There is many factor that simulateneously involved in AD. To check these you must go with antioxidant, AchE, LOX, Amyloid Protein cell line protection assay, serotin assay and many more in vitro assay.

To locate any small brain part, first you have to under stand the rat brain atlus in coronal sections. The main part on the coronal section is the varying size of the hippocampus. Hippocampus starts from the optic chiasmaa in small size and as the sections proceed to posterior it goes down and down and make a complete half ring or comma like structure on both side of the hemisphere. You may easily locate the position of the substantia nigra. The rats brain atlus and abbreviation used for the substantia nigra has been pointed out by Dr. Sushil Kumar.

Thanks Jeffrey. I need to sincerely think on your points.

Dear Marianne, we have been studying retinal diseases for a few decades and in the last decade, have developed the use of save, non-invasive therapies with no side effects reported. These are dietary saffron and near-infrared light therapy. See my publications for example. I'm under Prof Jonathan Stone's group at Sydney Uni and he is the brains behind these studies using saffron and infrared light therapy for AMD, retinitis pigmentosa, Parkinson's disease and Alzheimer's disease. These therapies are said to be protective by reducing oxidative damage and switching on mitochondrial protective pathways.

Permissive hypercapnia may induce augmented cerebral blood flow in the situation of relative low-perfusion pressure after resuscitation from cardiac arrest could have

beneficial effects to the globally ischemic brain.

Hi Natascha, I use homemade buffers for running and transfer, the lysisbuffer is a simple Tris-EDTA-TritonX mixture (pH7,5) with proteaseinhibitor cocktail from Sigma (you can find the lysisbuffer recipe on the Abcam homepage). My antibody is from Cell Signalling LC3B (D11). Check out the publication in Autophagy 3:6, 542-545; November/December 2007 about how to interpret LC3 Immunoblotting, it is very interesting. Make sure that your protein hasn´t travelled too far (prestained marker - any company will send you a testsample, if you do not have any at hand) and maybe try another antibody (other company). I would also give it a try with a high percentage homemade gel, Valeria also uses 13-15% and it is not that much work to pour a gel. What about your transfer, can you detect the marker on your film and is your stack packed tightly (I once had a smear when I didn´t have a tightly packed stack), do you check transferefficiency with a reversible stain? (I use MemCode from Thermofisher) What do you use for blocking, I blocked once with milkpowder and did not get any signal from that blot and do you expose for a long enough time, one of my blots gave good signals only after 8 hours of incubation!

It depends on how you are going to use your estradiol and for what purpose. As long as estradiol is able to form hydrogen bonds with your solvent, you should be able to use it but keep in mind that the solvent will stay in your prep.

HT-22 (mouse hippocampal neurons) seems to be capable of expressing estrogen receptors as evidenced by:

"In order to obtain data regarding the interaction between RyR and ERβ, we examined co-localization patterns of both proteins in intracellular compartments in the neuronal cell line HT-22. While initially thought to be free of functional endogenous ERs [45], the HT-22 cells were found in recent studies to express both endogenous ERβ [30] and ERα [46]".

ref: Rybalchenko V, Grillo MA, Gastinger MJ, Rybalchenko N, Payne AJ, Koulen P. The unliganded long isoform of estrogen receptor beta stimulates brain ryanodine receptor single channel activity alongside with cytosolic Ca2+. J Recept Signal Transduct Res. 2009 Dec;29(6):326-41. doi: 10.3109/10799890903295168.

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Affiliation: SOFA (Sarcopenia & Osteoporosis Fraction in Ageing)

The answer to the question “Do you recommend anything besides lowering intraocular pressure for your glaucoma patients” is complex. Glaucoma is a misunderstood disease. Until we fully understand the disease we can’t cure it. Even the role of lowering of IOP is questionable especially in NTG, why to worry about adjuncts to lowering of IOP until we fully understand this disease. We can’t treat a disease unless we fully understand it. In glaucoma we don’t even know what actually is happening to the nerve fibers. Are the nerve fibers really being atrophied in glaucoma? All we hear is that nerve fibers are being damaged in glaucoma. But word damage is very broad. What kind of damage we are talking about? Are the nerve fibers being compressed, crushed, sliced, severed or anything else. With all the recent technology we even can’t answer with certainty above basic question of glaucoma.

Lowering of the IOP in glaucoma may be slowing the disease at its best but surely unable to halt it. Therefore, it appears we are following a wrong path in glaucoma. We are following a 150 –year old paradigm of cupping without even fully understanding the phenomenon of cupping or excavation occurring in the glaucomatous disc. Ironically, we are using the term cupping to describe both physiological as well as pathological cupping. It is unheard of using the same parameter in describing both the healthy and disease state of an organ in other branches of medicine as we do in glaucoma. No wonder why the term cupping and cup/disc ratio has produced a conundrum in the glaucoma diagnosis.

Glaucoma is defined as optic neuropathy implying that nerve fibers are being atrophied in glaucoma. Optic neuropathy is big umbrella term which includes hundreds of diseases in which optic atrophy is occurring. Is glaucoma also an optic neuropathy? We must not ignore the very important fact that glaucomatous field defects reveal that nerve fibers are always being destroyed in a specific sequence starting with peripheral fibers, then arcuate and ending with central. It is unlikely that raised IOP acting directly or via ischemia would always cause atrophy of nerve fibers in a specific pattern and not destroy the nerve fibers haphazardly. If we believe that RGC’s are the primary site of injury in glaucoma then this scenario becomes even more puzzling. How is it possible that raised IOP or in fact any pathology directly or indirectly will always destroy systematically those RGC”s first which serve the peripheral vision and not occur randomly? I have been puzzled by aforementioned question in my entire professional career spanning over 50 years. But now I may have answer to this puzzling question at least to my satisfaction.

I learned very interesting things by comparing the morphology and histology of the glaucomatous disc with the non-glaucomatous optic atrophy such as due to multiple sclerosis. Morphology and histology of the glaucomatous and non-glaucomatous optic atrophy are distinctively different. Glaucomatous disc is excavated whereas the non-glaucomatous atrophic disc is non-excavated and flat. Blood vessels are sloping/ kinking at the disc margin in glaucomatous disc but not in the non-glaucomatous optic atrohies. Wedge shaped defects in the retina are present in glaucoma but not in non-glaucomatous optic atrophies. Histology of end-stage glaucomatous disc resembles an empty bean-pot devoid of nerve fibers, whereas in non-glaucomatous optic atrophies the nerve fibers though shrunken are still present.

Sloping/kinking of blood vessels at disc margin in the glaucomatous disc, prior to any change in contour of physiological cup, suggests that optic disc may be sinking in glaucoma. Excavation or empty spaces occurring in the glaucomatous disc can only be explained due to severance of the nerve fibers, not due to their atrophy. End-stage glaucomatous disc may not be 100% cupped atrophic disc but a crater left over after severance of all nerve fibers. Wedge shaped defects in the retina and arcuate field defects in glaucoma can only be explained by severance of the arcuate fibers, not by their atrophy. Progressive thinning of the RNFL can only be explained by continuous severing of the nerve fibers in the glaucomatous disc and it is not occurring in non-glaucomatous optic atrophies. In view of aforementioned I hypothesize that nerve fibers are being severed in glaucoma due to sinking disc. If true, then glaucoma is in fact a herniation of the disc in the scleral canal, a mechanical failure. In this regard the lowering of IOP may be too late. Lowering of IOP may serve as a prophylactic treatment but not an ultimate treatment if disc has started sinking and glaucoma has been initiated. We may have to find ways to stop the further sinking of the disc. Analogy: statins would prevent the build up of plaques but if the plaques have already formed then cardiologists would either break the plaques or bypass them by surgery. Thank you.

there is little more add. You need to take your compound's formula and synthesis to a radiochemist. They can advice you on potential pathways.

11C has only a half lifetime of 20min. you can analzye it for a few half life times. Nice thing is that if you compound comes with methylated groups, it is a natural fit.

Talk to a chemist on this one with your data, formula etc. We have hit about the limit on how to go forward without knowing more specifics.

If you employ radio methods, you will discover how powerful they are as a tool. It is almost magic what you can do compared to traditional methods for tracing compounds due to the extreme sensitivity and ease of detection.

There is a lot of infrastructure required, but there are experts and labs around to support you.

Best

Marko

Hi Nat. I agree with Yannick that the main problem with AD models is their lack of neuronal death. However, if you first want to check that your drugs work by reducing amyloid burden, you can use any of the AD models. 5xFAD animals are ok to have quick preliminary results since their pathology stars around 1.5 months, althogh sometimes it's a model too aggresive to be able to see any difference. You can also try with double APP/PS1 (pathology at 6 months) or Tg2576 animals (9-12 months). As suggested before, 3xTgAD animals are a good option because they have some cell death. We have all of these animal models and also type 2 diabetes and prion animals, just in case you want to check whether your drugs also work in other protein misfolding disorders. Best, Ines.

Actually my thesis work concluded that brainstem auditory evoked potential are affected significantly in type 2 diabetes irespective of current blood sugar level / disease duration. now i am planning to do research on topic of question. but i am not getting any good research on this topic. what do you suggest sir?

I suggest you do not use them. I have performed neurotoxicity experiments on human neuroblastoma SH-SY5Y cells (PC-12 are from rat and more transformed respect to SH-SY5Y) then I have repeated the experiments on rat embryonic DRG neurons and results have been different. Rat embryonic DRG naeurons are a very good model to study neurotoxicity.

Good luck.

Gabriella

If you have a Speedivac or like machine, that will do the job without too much trouble. You can then resuspend in your media of choice to desired volume.

Thank you sir for your answer. I would be very happy if you can provide some published references regarding the neuroprotective effect of Haritaki against glutamate induced toxicity...

In this article the authors do 30 min exposure for ERK and 60 for CREB to several NMDA concentrations.

Still, I'd say it may differ if you're working with slices instead of cultures. Hope it helps!