Inner Ear Hair Cell Regeneration — Maybe We Can Know More

[Jim51042]

No. Read the paper.

Sorry. Wow awesome I am a lot more enthusiastic!!!

 

[Samir]

I didn't.

Interesting...

Is it the basilar membrane that prevents them from migrating into the cochlear duct?

When they say that they inject the cells into the scala media, do they enter via the scala tympani, through the basilar membrane?

This must be so frustrating for them! They need to reach the Dieter cells and the tunnel of Corti, but if they release the cells in scala tympani they won't migrate. How could they? They can't diffuse across the basilar membrane, can they? Yet if they release them in the scala media they are killed off by the endolymph.

Consequently, it is necessary to inject the cells directly into the scala media, the fluid-filled lumen within the cochlear duct epithelium.

But where in the scala media do they release the cells? And how do they enter the scala media? I can't figure that out from the text. Correct me if I am wrong but only the topical portion of the epithelium, i.e. the cilia is in direct contact with endolymph. The cell bodies are not, otherwise they would all die and collapse.

So I am thinking, can they not inject the cells directly into the epithelium, rather than into the potassium rich endolymph in scala media? Because if they just release them into the endolymph hoping that they will land on top of the epithelium and integrate, I would not expect them to survive.

 

[Samir]

They are injected through the RWM or oval windows (or diffuse across the RWM) into the perilymph.

How does oval window delivery work? They remove the stapes bone?

the virus migrates throughout the inner ear

I think this is because the virus is released into the endolymph fluid, and it flows with the fluid, lands on the epithelium, sinks in and delivers the payload which starts to integrate with the supporting cells. The most successful of these so far, the ANC80L65 is specifically designed to withstand the potassium rich environment.

But if they were to inject the virus directly into the epithelium, I don't think it would be very successful. The virus would not be able to spread throughout the entire length of the epithelium. We would probably see very localized transduction.

 

[Aaron123]

Is it the basilar membrane that prevents them from migrating into the cochlear duct?

I believe so.

When they say that they inject the cells into the scala media, do they enter via the scala tympani, through the basilar membrane?

My understanding is that they drill a hole in the cochlea and inject directly into the scala tympani. This may vary by paper, but from http://www.nature.com/articles/srep46058

"Using the dark stria vascularis as a landmark, scala media was opened by cochleostomies in the second turn and third turn. Experimental substances where introduced through the second turn cochleostomy and excessive fluid was allowed to escape from the third turn. First, 2 μl of stem cell media (TeSR-E8 supplemented with 10 μM Y-27632, see stem cell procedures) or media with 10 mmol/l sodium caprate (Sodium decanoate, Sigma-Aldrich Co., St. Lois. MO) were injected into scala media. As noted above, some animals received only these fluids to assess their effects on native tissues, in others, these fluids were followed by 2 μl of human stem cells (~20 million/ml)."

 

[Samir]

I get that this is a amazing leap forward in terms of hearing restoration but using up your supporting cells in this procedure is risky especially you don't have a lot of hearing loss.

Why would it be risky?

You lose some, you win some. I understand your concern, but it's not like you lose all your supporting cells. The cells that Frequency is targeting are the progenitor cells. This is a sub-set of the supporting cells.

My question is if a newly grown hair doesn't get connected up does it die.

Why would it not get connected?

I don't think it would die. But it would not help you hear better.

 

[Aaron123]

How does oval window delivery work? They remove the stapes bone?

I believe they drill a hole it it. This is the procedure for the Genvec/Novartis trial.

lands on the epithelium, sinks in and delivers the payload which starts to integrate with the supporting cells

This seems plausible.

The most successful of these so far, the ANC80L65 is specifically designed to withstand the potassium rich environment.

I've seen no evidence that Anc80 is designed with potassium in mind. It's not mentioned in the methods paper (http://www.nature.com/nbt/journal/v35/n3/full/nbt.3781.html). It's also not necessary as the injection is through the RWM into the perilymph (see methods section of the paper). In fact, in the paper, they speculate "The reason for lack of rescue in 6 of 25 mice was not clear, but may have been due to failed injections, clogged injection pipettes or injections that missed the targeted perilymphatic space." So Anc80 is not injected into a high potassium environment.

The virus would not be able to spread throughout the entire length of the epithelium.

Again, this seems plausible though I don't think I have seem specific discussion of why it is that Anc80 is more effective across the full range of IHCs.

 

[Samir]

My understanding is that they drill a hole in the cochlea and inject directly into the scala tympani.

"Using the dark stria vascularis as a landmark, scala media was opened by cochleostomies in the second turn and third turn."

This might explain why it was not very successful. Cells don't survive as well as viruses. I really think they could have better success by injecting the cells into the epithelium, instead of just releasing them into the fluid, crossing their fingers and hoping for the best. Most viruses don't perform this task well, how could these cells? How they would reach the epithelium for direct injection I do not know, but they will have to come in from the scala tympani or scala media.

Making a hole in stria vascularis seems risky to me. It is responsible for the production of endolymph.

I believe they drill a hole it it. This is the procedure for the Genvec/Novartis trial.

I assume this is the same type of procedure they use for stapedotomy when stapes footplate gets stuck. I read somewhere that they use 0.7 mm microdrill or laser to make the hole. But in stapedotomy and stapedectomy, they don't drill the hole all the way through.

How do they seal the hole? Do they use bone cement? Like in the study you linked to?

"A piece of muscle was placed over the cochlea opening and the bulla was closed using bone cement."

http://www.nature.com/articles/srep46058

I can see why Auris, and many others are hoping that RWM diffusion will be good enough for therapy. All these other approaches are very risky.

Again, this seems plausible though I don't think I have seem specific discussion of why it is that Anc80 is more effective across the full range of IHCs.

I am just speculating on potassium resistance. I don't have full access to that article.

So stem cells delivered to scala tympani don't migrate or diffuse across the basilar membrane effectively. But viruses seem to do this well. So what is it then that makes viruses so effective? Especially since they are delivered to scala tympani, and not scala media? I would expect better transduction when delivered to the scala media. I believe this is the approach professor Stefan Heller is working on.

Can we use viral vectors to deliver stem cells?

Where does this leave molecular approaches like the ones that Frequency is developing? Are they potassium compatible? Can we use viral vectors for these molecules?

 

[Jim51042]

For those with a good sense of history outside of GenVec how many hearing restoration human clinic trials have there been? Kinda seems like a good sign that more than 5 are gearing up.

 

[Jim51042]

I can see why Auris, and many others are hoping that RWM diffusion will be good enough for therapy. All these other approaches are very risky.

For someone with access to the RWN article above do you know how it would compare with the staple approach in terms of the percent of OHC and IHC it reaches. Either way with all the testing that has occurred I think ANC80 with the coating is ready for human testing.

 

[Aaron123]

I believe this is the approach professor Stefan Heller is working on.

What papers do you have in mind?

Can we use viral vectors to deliver stem cells?

No.

Are they potassium compatible?

Doesn't matter. They diffuse through the RWM into the perilymph.

Can we use viral vectors for these molecules?

No.

 

[GregCA]

I assume this is the same type of procedure they use for stapedotomy when stapes footplate gets stuck. I read somewhere that they use 0.7 mm microdrill or laser to make the hole. But in stapedotomy and stapedectomy, they don't drill the hole all the way through.

They do. How else is the prosthesis going to act as a piston and move freely in the hole?
There's only a sleeve that is inserted between the piston and the hole, which is made from a graft. For me they took a vein from my hand.
The piston has to be able to transmit the mechanical vibrations from the ossicular chain into fluid waves in the cochlea.

That's actually one of the risks with the procedure. For people who have endolymphatic hydrops, there can be extra pressure from the fluid in the cochlea which results in a "gusher" when they finally poke through. It's not good when that happens.

 

[Reinier]

Reading all the posts and some documents (very basic understanding) lately I am starting to think Gen-therapy will be suited better for inner ear regeneration than stem cell therapies. At this moment in time that is. Perhaps in future that will change?
I used to think that stem cell therapies would be first.

 

[Samir]

They do. How else is the prosthesis going to act as a piston and move freely in the hole?

What kind of procedure did you have? Did they remove your stapes arch and footplate?

I have read about this earlier. My understanding is that they don't drill the hole all the way through. They just drill enough so that they can anchor the piston in the hole. The hole is 0.7 mm, and the piston is 0.6 mm. I don't know how deep the hole is, but if I remember correctly they stop the drill about 1/2 mm away from entering the fluid filled space.

Stapedotomy:


Stapedectomy:

There's only a sleeve that is inserted between the piston and the hole, which is made from a graft.

That sounds like you had stapedectomy?

For me they took a vein from my hand.

Vein from your hand? I'm not sure what that means. Could you explain?

The piston has to be able to transmit the mechanical vibrations from the ossicular chain into fluid waves in the cochlea.

It will do that, the piston becomes part of that chain. But I don't think need an open hole in the bony labyrinth. You just need to shake the footplate at the oval window. This may be done a little differently, but I am pretty sure they don't drill a hole all the way through. What good would that be? The piston would just move in and out. It needs to grab hold of the footplate, if it's still present, or the surgical graft and shake it back and forth.

That's actually one of the risks with the procedure. For people who have endolymphatic hydrops, there can be extra pressure from the fluid in the cochlea which results in a "gusher" when they finally poke through. It's not good when that happens.

I still don't think they intentionally want to or need to poke a hole all the way through. What you describe may be the result of a partially failed procedure, where they just went in too much. They need to stop drilling 0.5 mm before they poke through. That's not so easy to achieve, it's a microsurgery. So I am thinking that they might have gone a little bit too deep.

Of course, I could be missing some important key fact here. I am no ENT surgeon! It's just something I have read about briefly, these different surgeries of the middle ear.

 

[GregCA]

What kind of procedure did you have? Did they remove your stapes arch and footplate?

I had a stapedotomy. They removed the arch but kept the footplate in place (they drilled through it).

That sounds like you had stapedectomy?

Stapedotomy. Pretty much everyone does stapedotomies now.

Vein from your hand? I'm not sure what that means. Could you explain?

They need some tissue from the patient to wrap the piston into before it goes into the inner ear. It has to be solid enough that it won't break (in years of friction), but flexible enough that the piston can move with it around it. It turns out that our veins are made of a tissue that is pretty good for that, so they opened my hand, cut a piece of vein, sliced it (the tube/pipe) such that they end up with a somewhat square piece, which is then used to wrap the prosthesis.

It will do that, the piston becomes part of that chain. But I don't think need an open hole in the bony labyrinth. You just need to shake the footplate at the oval window. This may be done a little differently, but I am pretty sure they don't drill a hole all the way through. What good would that be? The piston would just move in and out. It needs to grab hold of the footplate, if it's still present, or the surgical graft and shake it back and forth.

My understanding is that it goes all the way through (hence the risk of gusher). I don't think they can just try to shake the footplate, because that's the very part that is ankylosed from the bone remodeling focus.
I think the graft acts as a new membrane to propagate the sound into the inner ear.
Watch this. It's actually described by the surgeon who did my surgery. It also shows the vein graft process.

 

[Samir]

@GregCA Thanks for sharing! I know this thread is about hair cells and nerves and such, but this is very interesting too!

How was your hand after this? Did it heal up nicely? It's bad that they have to cut your arm open for this. It would be nice if they could come up with some biomaterial that does the same job.

How was your hearing after the surgery? How much did it improve?

What is it about otosclerosis that causes tinnitus? It can't possibly damage the hair cells? I just found this explanation:

The conductive type of otosclerosis usually progresses up to a maximum when patients are in their 30s. After this, it rarely progresses. Dizziness or imbalance is a feature of otosclerosis in roughly 25% of cases. Between 40% and 65% of patients have tinnitus (ringing in the ears) (Gristwood & Venables 2003, Sobrinho et al 2004). In about half of all patients, there is a family history of similar problems. Tinnitus appears to improve following surgical intervention in a number of cases, since "the conductive hearing deprivation produced by otosclerosis is associated with limited and reversible modifications in the central auditory pathway that are linked to tinnitus"(Deggouj et al 2009).

http://american-hearing.org/disorders/otosclerosis/

This text refers to "central auditory pathway". I'm not sure why they say "central" pathway. Other studies have shown that conductive hearing problems can trigger neural behavior changes in the early stages of auditory pathway, at the cochlea. It also says that it's "limited and reversible modifications". Did your tinnitus decrease after the surgery?

 

[Aaron123]

If you guys want to have an in-depth discussion about stapedectomy/stapedotomy, it would make sense to start a new thread (unless there is an existing one).

 

[Jim51042]

I am starting to think Gen-therapy will be suited better for inner ear regeneration than stem cell therapies. At this moment in time that is.

What made you come to this decision? The paper describing the toxicity of the potassium channel? I wonder if the gel used in these viral injection would help with the survival of injected stem cells.

 

[GregCA]

@GregCA Thanks for sharing! I know this thread is about hair cells and nerves and such, but this is very interesting too!

Yes, the mods should feel free to move the last couple of messages to their own "Stapedotomy details" thread.

How was your hand after this? Did it heal up nicely?

Actually no, it got infected. It almost never happens, but hey, almost never is not never. Topical antibiotic helped healing. That's nothing, it's just superficial. I'd happily trade my T for hundreds of these over my body. Now it's just a scar on my hand.

How was your hearing after the surgery? How much did it improve?

I had mixed loss going in (conductive + sensorineural). The surgery pretty much closed the air-bone gap completely, so I did regain significant hearing in the low frequencies. However, my high frequencies got worse (which is a known risk). That's the only negative thing out of the surgery: I didn't get much dizziness, didn't lose taste or get any facial problems (they didn't have to touch the chorda tympani), etc.
The recovery is a bit difficult because of the packing in the ear: you can't hear anything for a while, and the T becomes really overwhelming.

I'm now left with sensorineural losses: it's all downhill from 1 kHz up.

What is it about otosclerosis that causes tinnitus? It can't possibly damage the hair cells?

It can, unfortunately (hence the sensorineural part of the losses). In some cases the bone remodeling process releases cytokines into the inner ear fluid, and they are harmful to the hair cells (see, we stay on topic here!). So if your otosclerosis starts attacking the inner ear, you can get SNHL. Look up "cochlear otosclerosis".

So the T can be a combination of individual Ts coming from various sources. The T that comes from the middle ear stiffness is generally low frequency. There is a paper that explains the fluid dynamics equations behind it - I read it about a year ago. You may be able to find it on pubmed (I think that's where I found it first).
The T that comes from the inner ear damage is more on the high frequency side, probably because the bone focus is close to the base of the cochlea, which is where the HF cells are (that's only my guess - nobody really knows).

I went into surgery with a 50/50 chance of getting rid of T. That was one of my main motivators. It didn't work for me. The T morphed into something different, with less of a pulsatile component. It's still horrible, but it's different.
The good news is that my severe hyperacusis got reduced to almost nothing after surgery. So that's a big win for me.

 

[GregCA]

If you guys want to have an in-depth discussion about stapedectomy/stapedotomy, it would make sense to start a new thread (unless there is an existing one).

Agreed. I think I gave all the info I had to @Samir in my last message. I don't expect a lot of follow up on that, but if there is we should definitely do that somewhere else.

 

[Samir]

Let me make it up to you for stealing the show, by introducing an outer hair cell from the apical part of the cochlea to the dance floor. Turn the volume down!



This was recorded on 13 August 1987 in England for a BBC show called Ear We Go.

This outer hair cell gets thinner when it gets longer and fatter when it gets shorter. Measuring up these changes indicates that the cell volume stays constant. This supports the idea that the 'motor' is a molecule whose job it is to change membrane area. The molecule, discovered in 2000 by Peter Dallos' lab in the US, is called 'prestin'.

https://www.ucl.ac.uk/ear/research/ashmorelab

 

[Jim51042]

@Aaron123 Can you discuss how the paper describing injected stem cell death within the ear matches some of the prior success Dr. Rivolta has had with neuron stem cells? Would neuronal stem cell for nerve repair and synaptic regrowth have to travel through the high K environment? Did he just lose a lot of stem cells and make up for that in terms of quantity?

https://www.tinnitustalk.com/thread...m-cells-into-the-inner-ear.17306/#post-201751

 

[Jim51042]

Can anyone speculate on the effects of multiple viral injections of the AAV or ANC-80? Would the immune system react to a second injection? Has this been tried in animals? I think multiple viral introductions may be useful for companies with the goal of increased synaptic connections using growth factors.

 

[Aaron123]

Can you discuss how the paper describing injected stem cell death within the ear matches some of the prior success Dr. Rivolta has had with neuron stem cells? Would neuronal stem cell for nerve repair and synaptic regrowth have to travel through the high K environment? Did he just lose a lot of stem cells and make up for that in terms of quantity?

Since the target was spiral ganglion neurons, the cells were not injected into the inner ear so potassium was not an issue.

Nonetheless, I am far more skeptical about this paper than many people here.

Can anyone speculate on the effects of multiple viral injections of the AAV or ANC-80? Would the immune system react to a second injection? Has this been tried in animals?

We could all speculate, but it would not, as far as I know, be based on any published research and so there would be little or no point.

 

[Jim51042]

Nonetheless, I am far more skeptical about this paper than many people here.

What makes you say that? I dont think your wrong because stem tech has been around long enough that more of these questions should have been answered. I know the US has stifled research. Still if you have lost or damaged beyond repair some SGNs would there be another to replace them? If gene or drug therapy is as successful as we hope there still may be utility in a success stem treatment.

 

[Samir]

I dont think your wrong because stem tech has been around long enough that more of these questions should have been answered.

What exactly are those questions?

I know the US has stifled research.

Don't worry, the US is not the only one researching this. Besides, didn't Trump appoint that pro-biotech guy as the head of FDA? He is for progressive approval of new drugs. That's a good thing, I think.

https://www.forbes.com/sites/patric...-progressive-approval-for-drugs/#2e7f45c46a7b

Still if you have lost or damaged beyond repair some SGNs would there be another to replace them?

Type 1 or type 2? If you can generate new ones, then there should not be a problem. If you can't, then it's probably more advantageous to damage Type 1 neurons if your main problem is bad hearing, or Type 2 neurons if your main problem is tinnitus. Type 1 is likely more responsible for tinnitus than Type 2.

"Type I (blue) spiral ganglion neurons (95% of the ganglion neurons) have a single ending radially connected to IHCs."

Type II (green) small, unmyelinated neurons spiral basally after entering the organ of Corti and branch to connect about ten OHCs, generally in the same row.

Scheme from Liberman

http://www.cochlea.eu/en/cochlea/organ-of-corti/innervation

 

[Samir]

This is what causes tinnitus in many:



"Acquired hearing loss is often accompanied by tinnitus. Tinnitus is an auditory perception (such as a whistling or hissing) without an external stimulus. This perception can be very debilitating and nearly 50% of all adult visits to otolaryngologists relate to tinnitus!

Some are simply internal sounds (caused by blood flow, jaw motion…) transmitted to the ear, but many are caused by a problem in the inner ear (cochlea), which makes treatment particularly difficult.

Recent research has focussed on a mechanism that could explain the origin of a large number of tinnitus cases: the disruption of the connections (known as synapses) between the sensory cells and the auditory nerve, which causes the auditory nerve fibres to be automatically activated with repetitive signals.

The traumatic sound (a very intense noise) damages the connection between the hair cell and the auditory nerve.

The auditory nerve fibre, missing the normal input from its partner, starts to self-excite, sending non-stop messages to the brain which are perceived as a whistling noise: this is tinnitus."

http://www.cochlea.org/en/impairment/tinnitus

This perfectly describes what I think is my cause of tinnitus.

 

[Jim51042]

Don't worry, the US is not the only one researching this. Besides, didn't Trump appoint that pro-biotech guy as the head of FDA? He is for progressive approval of new drugs. That's a good thing, I think.

Actually they went another way with this see article below. So we will still have Phase 2 and Phase 3 before market release

http://www.npr.org/sections/health-...gottlieb-to-head-food-and-drug-administration

 

[Samir]

@Jim51042 So they went with Scott Gottlieb instead of Jim O'Neill? Anyway! Don't worry too much about that. Like I said, the US is not the only one interested in this. We have scientists all around the world doing some pretty amazing stuff. Everyone is interested in inner ear regeneration these days. One way or another we will get there.

What I would like to see is proof of concept in a lab. To see that the treatments actually work. At least on animals, for now.

 

[Reinier]

What made you come to this decision?

Apparently it is difficult if not impossible at this moment in time to get stem cells to survive in the inner ear.
Also the first attempt (GenVec) is a gen therapy. The one Frequency Therapeutics is discussing is also gen therapy.

 

[Jim51042]

Apparently it is difficult if not impossible at this moment in time to get stem cells to survive in the inner ear.

Do they have to get to the inner ear to be effect for neuron regrowth? Could/do the majority of stem cells injected into the middle ear make it to the SGN? If they do how much type 1 and type 2 neuronal regrow results? If at this time stem cell injected into the middle ear are not useful for hair cell regrowth but do create type 1/2 nerve fibers then for tinnitus that is the whole ball game and we should be happy that there is a different treatment option. If not than I agree stem cell are a long way off from being useful for hearing restoration.

@Samir I know. But in the US I kinda feel like DC is tying the hands of these research facilities.