the sessions take place 5 days per week with a duration of 1hour delivering 600 contractions .
this is how we deliver the paradigm : basically , the rats are restrained in a plastic tube , in which they are free to move .
you can actually see what these contractions here look like  once every 6 seconds .
so , here is what we did to test this paradigm . what happens if we use this paradigm after nerve transection and stimulate the muscle for two weeks ?
one group had the right hind limb stimulated for two weeks starting 2 days after surgery .
we made two incisions : one for nerve transection and one to implant the electrodes .
the two parameters we looked for were motor unit number estimation ( mune ) and immunohistochemistry for endplate staining . for mune
there are two ways to estimate it : by electromyography ( emg ) or by muscle force .
what we did was to connect the stimulator proximal to the injury and then two electrodes in the muscle .
then we can calculate the mu count by dividing the maximum response by the average response from the 15 - 20 templates .
after two weeks the motor unit numbers in the muscle were significantly higher , a more than 2-fold increase ( figure 1 ) .
when you look at the emg you can see that there is a more robust emg . clearly , there is more reinnervation taking place . by immunohistochemistry ,
the endplates may be stained in red , while in green may be labelled the axons .
it is pretty clear that the stimulated muscles contained greater numbers of innervated endplates and intramuscular axons .
they showed a greater number of axons in the distal stump of stimulated muscle . observing a nerve section from a rat that did not receive es , proximal to be nerve injury
all the axon appeared intact while at the injury site , even at high resolution , the site is pretty dark , which means there were not many axons growing .
in contrast , in a stimulated rat at the site of injury there were many more axons at both high and low resolution .
it was pretty clear that our muscle stimulation paradigm of early stimulation works and increases motor reinnervation .
a further question is : what happens in the long run if we keep stimulating throughout the reinnervation period ?
the answer is a next study , in which es lasted for 1 , 2 or 3 months .
we also looked at functional metrics with the walking track analysis and tapered beam tests .
we divided them into 5 categories : normal reinnervation , reinnervated by a nodal sprout , reinnervated by a terminal sprout , reinnervated but unknown ( we do nt know where the axon was from ) and of course there were denervated endplates .
in fact , if you combine all those reinnervated only 10% of the muscle endplates were denervated , the rest were reinnervated .
so , it might be that electrical stimulation reduces terminal sprouting , we could say this is bad , but look at the percentage : it s only 5% ( figure 3 ) .
the next thing we analyzed was the functional metrics over 3 months . every week , for three months we took behavioral tests to observe functional recovery .
the walking track analysis was for measuring overground locomotion and the tapered beam test for measuring skilled locomotion . for the walking track test
we dip the rat s feet in ink and let them walk across a piece of paper .
what we had to measure were the toe spread , the print length and the intermediate toe spread and you plug these data into a formula and you get a number called the tibial function index .
we found out that it is not a really good metric for tibial nerve injuries .
we have full slips and half - slips , which allows us to calculate the slip percentage .
after 6 weeks we noticed the rats stopped using their backwards sliding motion they used before . finally , a video at 3 months showed that the unstimulated rats are still slipping a lot .
after week 4 there is a separation taking place between unstimulated and stimulated groups of rats . in summary it is clear proof that stimulation increases functional recovery and that he reinnervation is not adversely affected at the neuromuscular junction level .
the early enhancement of reinnervation can be potentially explained by evidence that stimulation of the proximal nerve stump following injury can increase the rate of axonal outgrowth across the injury site .
data from earlier this week on bdnf and gdnf show that both were significantly upregulated 2 weeks after injury ( figure 5 ) .
if we change the muscle , intensity and so on , can we achieve similar results ? also , we want to examine the distal stump factors during the first weeks of stimulation following nerve injury and repair .
what about the combination of proximal nerve stimulation during nerve repair with daily muscle stimulation ?
does muscle stimulation affect synkinesis ( nerve misdirection ) ? if you cut a nerve then the axons are not going to grow back to the original muscles .
we have the soleus and the lateral gastrocnemius and they are innervated by a common branch - the lateral gastrocnemius - soleus nerve .
we inject some blue dye into the soleus and this goes to the spinal cord after some time .
then we do a nerve transection and repair and connect the stimulator to the soleus .
after 2 months we cut that soleus nerve and we basically put the nerve in a well , for which we use red color . ideally , all the axons labeled by injection will be labeled when you dip this nerve in the well .
the idea behind all this is to take all these spinal cords and we take some sections and we quantify these labeled neurons .
we want to see where these motoneurons are and also we want to see if they come from the same population after injury .
audience question : how stable is the reinnervated phenotype if you stop the electrical stimulation ?
so far , it looks like there is , but it still needs some more research . audience question : we have heavy constraints from our surgeons because they are not open to stimulation after the surgery and during repair .
clinically , i can say that we had four pediatric patients that received nerve stimulation .
we did the stimulation on these patients during nerve repair for 1h and it was no problem .
audience question : have it been single twitches for the stimulation of the hind leg ?
kern : we have to take in account , if we stimulate seniors , we should avoid any negative effect on the regeneration process .
therefore , we invited mike willand . for me it is important that we avoid damage and that we do not influence the nerve regeneration in seniors .
so , i am very thankful and happy . when i saw this for the first time , it was not clear if the muscle stimulation was enhancing or inhibiting the axon regeneration . here
now we can be sure that the fes stimulation will not disturb the eventual nerve reinnervation process .
audience question : how stable is the reinnervated phenotype if you stop the electrical stimulation ?
so far , it looks like there is , but it still needs some more research . audience question : we have heavy constraints from our surgeons because they are not open to stimulation after the surgery and during repair .
clinically , i can say that we had four pediatric patients that received nerve stimulation .
we did the stimulation on these patients during nerve repair for 1h and it was no problem .
audience question : have it been single twitches for the stimulation of the hind leg ?
kern : we have to take in account , if we stimulate seniors , we should avoid any negative effect on the regeneration process .
therefore , we invited mike willand . for me it is important that we avoid damage and that we do not influence the nerve regeneration in seniors .
so , i am very thankful and happy . when i saw this for the first time , it was not clear if the muscle stimulation was enhancing or inhibiting the axon regeneration . here
now we can be sure that the fes stimulation will not disturb the eventual nerve reinnervation process .
 electrical muscle stimulation following peripheral nerve injury has been a controversial method of treatment due primarily to the inconsistent literature surrounding it . in this presentation transcript 
 i outline ongoing experiments investigating a clinically translatable daily muscle stimulation paradigm in rats following nerve injury . 
 results show that reinnervation of muscle and functional behavioural metrics are enhanced with daily stimulation with upregulation of intramuscular neurotrophic factors as a potential mechanism . 
 in addition , the impact of stimulation on terminal sprouting , a mentioned negative aspect of electrical muscle stimulation , was a minor contributor to long term functional reinnervation of stimulated muscles in our studies . 
A moderate ES paradigm
Assessing early reinnervation following ES
Assessing long-term reinnervation and functional recovery
Intramuscular neurotrophic factors
A rodent model to assess synkinetic reinnervation following daily ES
Questions from the audience and answers