peripheral nerve injuries are commonly caused by trauma to the upper limbs and are present in an estimated 23% of all patients admitted to a level 1 trauma center ( noble et al . , 1998 ) .
the economic impact of nerve injuries can be large with operative costs , hospital charges , rehabilitation visits , and lost time at work .
only subtle improvements to peripheral nerve repair have been made recently , and our current knowledge of nerve physiology and regeneration vastly exceeds our current repair capabilities .
poor outcomes of peripheral nerve injury are largely due to the slow process of axonal outgrowth . after nerve injury ,
severed proximal axons with intact cell bodies can grow up to 1mm / day . regenerating axons preferentially target appropriate end organ receptors but do not always take a direct route to their target ( fox et al . , 2012 ) .
thus , functional recovery is not always obtained due to slow growth and poor alignment of motor and sensory fibers .
muscle atrophy initiates immediately after muscle denervation , and if motor axons do not reach their target muscle within a critical time window , muscle tissue is less receptive to re - innervation . the most common current strategies to augment recovery after nerve injury such as decellularized nerve allografts , tissue matrices and nerve growth guides involve techniques to enhance axonal regeneration and decrease surrounding inflammation . despite these advancements ,
the combination of slow axonal outgrowth , wallerian degeneration , and muscle atrophy are still barriers to significant progress .
invertebrate axonal fusion after nerve injury is a natural mechanism that promotes rapid and highly specific recovery of neurons .
given the mechanism of axonal fusion is possible , peg has been investigated as an agent to promote axonal fusion in vertebrates .
bittner et al . pioneered initial studies using peg to fuse crayfish and giant earthworms axons with improvement in axonal fusion when axonal endings were exposed to a calcium free saline solution ( bittner et al .
the addition of an anti - oxidant such as methylene blue ( mb ) was noted to have an additional positive impact in rat sciatic nerve repair ( spaeth et al . ,
the proposed mechanism is peg - induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur . in an axonal injury without peg , axonal endings seal after an influx of calcium , preventing axonal fusion ( yoo et al . , 2003 ) .
figure 1 demonstrates the bioengineered process of peg fusion . when severed axonal endings are exposed to calcium - free hypotonic saline and an antioxidant ( i.e. , methylene blue or melatonin ) , vesicle - mediated sealing
peg is then applied to artificially induce closely apposed membranes of severed axonal ends to flow into each other and fuse .
this produces a partial repair of the plasmalemmal membranes that are then perfused with calcium containing saline , which causes vesicles to accumulate and seal remaining holes at the injury site .
mechanism of polyethylene glycol ( peg ) axonal fusion . reprinted from rodriguez - feo et al .
the success of the peg - fusion technique is based on multiple steps and factors .
peg , a hydrophilic compound , enhances axonal fusion in either severed or crushed settings and restores the ability to generate compound action potentials across the site of injury .
peg potentially facilitates lipid bilayer fusion by removing water molecules from the lipid bilayer at or near the damage site ( figure 1 ) .
sealing of axolemmal damage occurs through a calcium - dependent accumulation of membranous structures that interact with nearby undamaged membrane to form a plug . in severed nerves ,
this calcium - dependent system plugs the cut ends of axons preventing them from fusing with an adjacent axonal stump .
our studies have taken advantage of this calcium - dependent process by incubating in a calcium - free solution prior to nerve fusion , which prevents axolemmal sealing thereby enhancing peg based fusion ( sexton et al .
oxidative damage has also been shown to play a role in ischemia reperfusion injury after peripheral nerve injury .
methylene blue has been previously shown to slow axolemmal sealing and thereby enhance behavioral recovery after nerve injury ( spaeth et al . ,
methylene blue has shown a protective effect if applied in the correct sequence ( i.e. , after nerve severance and before peg based fusion ) .
taken together , these studies have shown that peg , with carefully controlled modification of the periaxonal environment can fuse simple nerve transections .
we have used the fusogenic properties of peg , the advantages of a calcium - free irrigation solution , and the antioxidant properties of methylene blue to demonstrate a rapid and decisive recovery of completely severed sciatic nerves in a commonly accepted mammalian model of peripheral nerve injury .
most current research efforts have focused on investigating peg fusion in a rat sciatic nerve model .
the rat sciatic nerve is easily accessible through a 23 cm incision posterior to the femur .
after a small cutdown to the nerve , the perineural tissue can be dissected easily to free the nerve for transection and repair .
our group has demonstrated the efficacy of peg fusion after neurotmesis with direct repair , nerve autografts , and nerve allografts .
compound action potentials ( cap ) are measurements of nerve conductivity and can be readily measured across healthy nerves . after nerve transection , caps can no longer be obtained due to axonal discontinuity .
caps are not restored after a direct suture repairdue to lack of axonal fusion secondary to axolemmal sealing after injury . however , after peg axonal fusion , caps are restored .
bittner et al . published cap data after direct suture repair with and without peg ( bittner et al . , 2012 ) .
baseline caps were obtained in all rats prior to nerve injury ( mean 4.1  0.16 mv ) .
postoperatively , caps were not measurable immediately after repair in the control group ( direct suture repair ) .
however , caps were restored within minutes after repair ( mean 3.3  0.23 mv ) in the peg fusion group .
, baseline caps were obtained in all rats prior to nerve injury ( mean 4.131  2.25 mv , n = 20 ) .
postoperatively , caps were not measurable immediately after repair or after 72 hours in the control group ( direct suture repair ) .
however , caps were restored immediately after repair ( mean 3.533  1.61 mv , n = 10 ) and after 72 hours ( mean 2.051  0.857 mv , n = 10 ) in the peg fusion group .
our experience with cap restoration in the peg fusion group was similar when using peg fused allografts ( riley et al . , 2015 ) .
our rat behavioral testing includes both the sciatic function index ( sfi ) and foot fault asymmetry score ( ff ) .
behavioral recovery was superior in the peg fused group in direct repair at 12 weeks ( bittner et al . , 2012 ) as well as the peg fused group in nerve allografts at 6 weeks ( riley et al .
our nerve autograft study evaluated rats up to 3 days , and the peg fused group already had superior behavioral outcomes ( sexton et al . , 2012 ) .
figure 2 demonstrates long - term behavioral outcomes after a standard microsurgical repair compared to a peg fusion repair after nerve transection .
when considering the electrophysiology and behavioral data together , it suggests at least a partial inhibition of wallerian degeneration after peg fusion .
behavioral assessments of sciatic nerve function according to the sciatic functional index ( a ) and foot - fault asymmetry test ( b ) .
sciatic function index ( sfi ) and foot fault asymmetry score ( ff ) scores ( mean  se ) were assessed from 3 days to 12 weeks post - operatively for two groups : control ( black dotted line ) and polyethylene glycol ( peg ) ( light - gray dotted line ) .
individual animals for all 12 weeks animals were graphed according to the aforementioned group color scheme ( data not yet published , manuscript in preparation ) .
after axonal injury , motor and sensory nerve counts are decreased distal to the injury site .
immunohistochemical staining using commercial antibodies against carbonic anhydrase ii ( ca2 ) and choline acetyletransferase is a method to stain for sensory and motor neurons respectively .
our research has shown increased sensory and motor axon counts in peg - treated nerve repairs ( figure 3 ) .
toluidine blue staining of peg fused nerves shows higher proportions of healthy myelinated axons compared to negative control nerves .
additionally , our lab has demonstrated the expression of npy1 , a marker of wallerian degeneration , in negative control nerves , whereas this is not expressed in peg - fused nerves .
these findings imply that we have delayed or even inhibited wallerian degeneration by fusing axons and maintained both axonal viability and function .
control ( a  c ) and polyethylene glycol ( peg ) ( d  f ) stained for choline acetyltransferase to label motor axons at 1 week ( a , d ) , 4 weeks ( b , e ) , and 12 weeks ( c , f ) post - operatively ( data not yet published , manuscript in preparation ) .
diffusion tensor imaging ( dti ) is a magnetic resonance imaging ( mri ) technique , which is an emerging diagnostic tool in nerve injury .
the institute of imaging science at our institution has developed a novel imaging protocol using dti for peripheral nerves . utilizing this protocol ,
we have been able to demonstrate the restoration of axonal continuity after peg fusion repair ( figure 4 ) .
representative tractography images show that axonal tracts do not extend more than a couple millimeters distal to the repair site in control nerves ( cut + suture , n = 6 ) . in peg
fused nerves ( n = 6 ) , the peg group had a statistically significant increase in the number of tracts that traveled through the repair site ( data not published , manuscript in progress ) .
our mri data supports the interpretation that peg - fused nerves establish axonal fusion which has been previously evaluated through behavioral testing and axon counts .
representative tractography of transected nerves harvested immediately following repair : control ( left ) and polyethylene glycol ( peg ) ( right ) .
green arrows indicate zone of repair ( data supplied by riley dc and kelm nd , data not yet published , manuscript in preparation ) .
despite the effectiveness we have observed with peg fusion , there can be inconsistencies in morphological and functional recovery of peg - fused animals . as demonstrated in figure 4 ,
peg fusion has not fully restored dti characteristics in these nerves , perhaps suggesting that not all axons were fused .
we speculate that a variability in the number of fused axons may in turn be responsible for the variabilities in morphological and functional recovery .
we also suspect factors such as the quality of repair , time of surgical intervention , and a surgeon 's level of experience with the technique play a critical role in achieving successful peg fusion .
our studies have included applying the peg solution manually through a syringe with the needle held at the surface of the neurorrhaphy .
this method , potentially , could result in small movements of the needle resulting in sub - optimal delivery of the peg solution or disturbance of the neurorrhaphy .
our current efforts in developing a device are focused on achieving a consistent nerve repair with uniform peg application to the neurorrhaphy site .
we recently described our experience with peg fusion using a nerve tube , which helps eliminate the need for sutures at the repair site ( sexton et al . , 2015 ) . our ultimate goal is to develop a simple coaptation system that will allow acute repair of severed nerves , with or without segmental injury , with minimal microsurgical training .
our lab has focused its efforts on small animal models , and the study of peg fusion has occurred across multiple laboratories and animal models . we have demonstrated repeated success in working in a rat sciatic nerve model .
bittner et al . has demonstrated the efficacy of peg fusion in invertebrate and small animal models in the aforementioned studies ( bittner et al . , 1986 ;
in addition , peg fusion has been investigated in large animal sciatic nerve and ex - vivo central nerve models in other laboratories demonstrating reproducibility of the technique ( donaldson et al . , 2002 ;
looking forward , we are currently conducting a phase i clinical trial of peg fusion in human nerve repair , and we were also hoping to move towards more large animal studies in the near future .
the ability to fuse severed nerves and regain rapid functional recovery could result in a paradigm shift in all aspects of patient care following peripheral nerve damage .
because this technique involves the use of current microsurgical techniques , with the simple addition of peg , it could rapidly be incorporated into a neurorrhaphy and autografting regimen .
our current clinical trial will investigate efficacy and safety of peg fusion in humans , and our development of a peg delivery device will make peg fusion reproducible with minimal training required to achieve superior results .
 the management of traumatic peripheral nerve injury remains a considerable concern for clinicians . with 
 minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury , outcomes of surgical intervention have been unpredictable . 
 the inability to manipulate the pathophysiology of nerve injury ( i.e. , wallerian degeneration ) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration ( ~1 mm / day ) . 
 when axons are severed , the endings undergo calcium - mediated plasmalemmal sealing , which limits the ability of the axon to be primarily repaired . polythethylene glycol ( peg ) in combination with a bioengineered process overcomes the inability to fuse axons . the mechanism for peg axonal fusion 
 is not clearly understood , but multiple studies have shown that a providing a calcium - free environment is essential to the process known as peg fusion . 
 the proposed mechanism is peg - induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur . 
 this review highlights peg fusion , its past and current studies , and future directions in peg fusion . 
Introduction
Polyethylene Glycol (PEG) Fusion Mechanism
None
Future Directions
Conclusion