Polyethylene Glycol Treatment for Peripheral Nerve Repair in Preclinical Models

Page 21 of 25 Polyethylene Glycol Treatment for Peripheral Nerve Repair in Preclinical Models Davis B. Rippee1, Gabriella E. Glassman2, Sara C. Chaker3, Patrick E. Assi4, Jennifer Black5, Alonda C. Pollins4, Jun Yao4, Wesley P. Thayer4,6* 1University of Mississippi School of Medicine, Jackson, MS 2Florida State University College of Medicine, Tallahassee, FL 3Vanderbilt University, Nashville, TN 4Department of Plastic Surgery, Vanderbilt University Medical Center, Nashville, TN 5Meharry Medical College, Nashville, TN 6Department of Orthopaedic Surgery, Vanderbilt University Medical Center, Nashville, TN 5Department of Psychology, University of Minnesota, Minneapolis, MN, USA 6Department of Neurology, University of Minnesota Medical School, Minneapolis, MN, USA


Introduction
Twenty million Americans suffer from peripheral nerve injuries each year; these injuries result in billions of dollars worth of healthcare expenses each year in the United States 1 . The vast majority of these injuries are due to trauma and they disproportionately affect those in careers that involve a high risk of traumatic injury. The most commonly affected demographic are males under the age of 40 and the most commonly damaged peripheral nerves include the ulnar, radial, and median nerves 2 .
Peripheral nerve injuries can have a devastating impact on a patient's quality of life. Symptoms include sensory and motor deficits that can result in paralysis of the affected limb and/or intractable neuropathic pain. Time is of the essence when treating these injuries, as muscle atrophy will ensue if repair is delayed.
Although there have been significant advancements in surgical technique and technology with regards to peripheral nerve repair over the past several decades, regeneration and recovery after these injuries remains problematic. Few patients with peripheral nerve injuries of the median and ulnar nerve achieve satisfactory motor recovery, and even less experience satisfactory sensory recovery 4 .
Many clinicians believe the predominant factor for the poor outcomes following these injuries is a failure to reestablish membrane continuity in the nerve 3 . This theory has led clinicians to seek out a variety of therapeutic measures that promote axonal fusion, including polyethylene glycol (PEG). PEG is produced by the interaction of ethylene oxide with water or ethylene glycol 5 . When combined with a calcium-free solution PEG can promote the fusion of axon plasma membranes and prevent the axon loss that occurs in Wallerian degeneration following peripheral nerve injury.
Discontinuity of the plasma membrane occurs during axonotmesis, and this discontinuity prevents the propagation of action potentials. Surrounding the nerve cell is a high extracellular calcium medium. When axons are damaged or severed, this extracellular calcium enters the peripheral nerve cell; this rush of calcium triggers the release of vesicles attached to the axonal microtubule system. These vesicles have a repair function and serve to seal the lipid bilayer, salvaging proximal axons and cell bodies. However, the distal axons undergo the Wallerian degeneration degradation process, resulting in distal axonal stump breakdown over the following week. This is why irrigation with a calcium-free solution is a critical component in PEG fusion-it prevents vesicle release and premature axonal stump fusion before cut axonal ends are mechanically reapproximated 7 . The addition of calciumfree solutions such as methylene blue cause the axon ends to remain patent, and the added PEG, hydrophilic in nature, helps remove water from the damaged lipid bilayer of the plasmalemma. This lowers the activation energy for plasmalemma fusion. Any remaining apertures are sealed with a calcium containing solution, which causes any vessels not previously activated to interact with the plasmalemma, sealing it from ionic transition 6 .
While several studies have confirmed the usefulness of PEG in treating peripheral nerve injuries, the comparative advantages and disadvantages of PEG compared to other therapeutic treatments have not been definitively shown in the literature. The overall goal of this systematic review is to provide a comprehensive evaluation of existing literature concerning the outcomes of PEG treatment on peripheral nerve regeneration in preclinical models.

Methods
A systematic review of literature was performed on articles published about preclinical studies involving PEG treatment of peripheral nerve injury. Data extraction was conducted on the results of articles meeting this review's inclusion criteria. Once the data was extracted, extensive analysis of the data was completed.

Search methods
The literature review was conducted under the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA, Figure 1). The MEDLINE database was queried using PubMed using the following keywords and phrases: "polyethylene glycol" OR "PEG" AND "nerve" AND "fusion".

Selection criteria
The inclusion criteria included articles that reported in vivo PEG treatment in animal models. After all of the articles were compiled, duplicate titles were removed. The two authors of this study, D.B.R. and S.C.C., then analyzed the remaining titles and abstracts. All included preclinical studies contained the treatment of a group with PEG after a peripheral nerve injury. Articles that published in a language other than English were excluded, as were any studies involving human research. Conference presentations and videos were not included in the criteria. Efforts were first made to determine the relevancy of the article from the abstract; if this could not be achieved, the full text was analyzed.

Data collection and analysis
Data extraction was performed by D.B.R. and S.C.C. during the spring of 2021. The following data measures were collected from the studies that met inclusion criteria: nerve type, lesion type, methods of evaluation, and outcomes. Nerve type referred to the specific peripheral nerve being treated; outcomes included morphological, functional, and electrophysiological. The advantages of using PEG as a treatment for peripheral nerve injury were compared to controls, and then the amount of significant findings in each of the data measures was evaluated.

Results
During the initial search of the literature, one hundred and seventy-nine articles were identified. After the application of inclusion and exclusion criteria, thirteen on animal models were identified. Only one of the studies utilized guinea pigs as the model, while the rest utilized rats. Ten examined the sciatic nerve, two involved the facial nerve, and one the femoral nerve. Distinctions were made for studies that evaluated peripheral nerve crush injuries versus cut injuries.
The results of the preclinical studies examined were evaluated through three different types of tests and/or recordings: motor, electrophysiological, and histological. The motor function was evaluated through the Sciatic Functional Index, or SFI. The SFI is a widely used metric in the study of the pathology of peripheral nerve injury. It is determined by comparing the geometric representation of the affected hind paw to the contralateral paw. Both are dipped in ink and the length and width of the print left by the rat as it walks across a piece of paper are measured 7 . In the nine of the thirteen studies that recorded this measure all except one reported an improve outcome 8 . This is likely most clinically relevant recording of the three so the fact that SFI improved after PEG treatment is indicative of its usefulness in PNI treatment.
Electrophysiological parameters were also measured in eleven of the thirteen studies. These measures included compound muscle action potential (CMAP) and force of muscle contraction. The restoration of compound muscle action potentials to normal levels and in turn force of muscle contraction is an indicator that PEG-mediated axonal fusion has been a success; the majority of studies reviewed showed that CMAP's were restored to normal levels.
Histological examinations are likely the most objective of the three categories of data analyzed. There were two main relevant histological outcomes encountered in the literature: number of axons in sites distal to the location of the peripheral nerve injury and the diameter of these axons. In the majority of studies, either one or both of these was reported to be improved 9-12 .
Of the thirteen preclinical studies analyzed, only Brown et. al 14 did not record PEG treatment having any sort of preferential outcomes compared to the control group. It is worth noting that this is one of only two studies that examined the facial nerve 14 . There are several possible explanations for this. This study conducted histological analysis on muscle rather than nerve and came to the conclusion that PEG treatment must not be useful for the facial nerve. However, analysis of their results and conclusion indicate that the study makes several Journal of Neurology & Neuromedicine study or series of animal studies that measured the posttreatment rate of survival of damaged Schwann cells would clarify this. Establishing a timeline for when PEG treatment is too late to be used clinically would also help set some guidelines. Bamba et al 13 reported successful fusion of a rat sciatic nerve twenty-four hours after nerve injury, which could be used as a benchmark upon which to further analyze the maximal feasible time gap in injuryto-intervention. Despite these mildly uncertain aspects, this systematic review shows the future of PEG nerve fusion as a viable clinical treatment of peripheral nerve injuries is bright.
Given the great success in animal models and low risk profile of the PEG substances, investigators have advocated for an early transition of PEG fusogen to human investigations 15 . To our knowledge Bamba et al 13 . were the first to successfully utilize PEG technology in human digital nerve injuries. The patients who received PEG had superior clinical outcomes and faster sensory recovery compared to a group of control patients with similar injuries 21 . We initiated a pilot study to evaluate the preliminary findings of PEG therapy in human upper extremities traumatic nerve injuries as well as identify any adverse events. Here we report our early repair findings in nerve recovery found in the first four patients enrolled. In this ongoing randomized control trial, we used PEG treatment for human peripheral nerve repair. The nerves repaired included two radial digital nerves, one median nerve, and one ulnar nerve. Results demonstrated meaningful or complete recovery in three of the four subjects 25 (Fig. 2). Additionally, an ulnar nerve repair at the wrist, and a radial digital nerve repair with an autograft, experienced meaningful recovery within four weeks, which is much earlier than typical expectations for recovery. This data demonstrates a proof of concept of PEG 3350KD can be used in human trials with no adverse responses.
assumptions; Bittner et al 17 concludes that the lack of peripheral nerve fusion in the study by Brown et al 14 . is likely due to the incorrect use of solutions 18 .
Two final clinically significant findings involved the actual process of applying the PEG treatment to the peripheral nerve. The first of these that should be noted is that methylene blue is the only solution that was shown to significantly enhance nerve fusion when applied before PEG treatment 15 . Other substances, such as methylprednisolone, melatonin, protein kinase C isozyme η pseudosubstrate fragment (ηPSF), and protein kinase A inhibitor (PKI), were almost completely unsuccessful [22][23][24][25] . In addition, Riley et. al 16 examined the use of a device that increases the consistency of PEG application. This device improved electrophysiological CMAP recovery by 12%.

Discussion
PEG therapy in the treatment of peripheral nerve injuries questions the idea that axon severance leads to the irreversible degeneration of the distal axon. The results of the preclinical studies examined in this systematic review of the literature show the future of PEG nerve fusion in clinical practice to be promising. However, there are still some areas to be reviewed. Almost all of the studies included measures of motor function recovery, but few examined any sort of metric for sensory recovery. Preclinical studies that review sensory recovery of the dermatomes covered by the damaged peripheral nerves would be helpful in establishing PEG treatment. Also, a more thorough investigation of autonomic nerve function after PEG treatment should be conducted, as the literature in this area is also lacking.
The effect of PEG on peripheral myelination, particularly Schwann cells, should also be investigated; none of these studies indicated whether PEG treatment of nerve injury prevents Schwann cell death. An animal Based on this systematic review and despite this limited case series, the use of PEG solution for PNI treatment is very promising with significant implications in humans. Future randomized clinical studies are needed to consolidate our findings. corresponds to a numerical value of 0, S1-S2 corresponds to 1, S3 to 2, S3+ to 3, and S4 to 4. A MRCC of S3, or 2, is denoted as meaningful clinical recovery.