Tacrolimus and Nerve Growth factor Treated Allografts for Neural

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Tacrolimus and Nerve Growth factor Treated Allografts for Neural Tissue Regeneration Yixia Yin, Gao Xiao, Kaiming Zhang, Guoliang Ying, Haixing Xu, Bruna A. G. De Melo, Shipu Li, Fang Liu, Ali K. Yetisen, and Nan Jiang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00452 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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ACS Chemical Neuroscience

Tacrolimus and Nerve Growth Factor Treated Allografts for Neural Tissue Regeneration Yixia Yin,a, b‡ Gao Xiao,c, e‡ Kaiming Zhang,d‡ Guoliang Ying,b, f Haixing Xu,a Bruna A. G. De Melo,b Shipu Li,a Fang Liu,d Ali K. Yetisen,g Nan Jiangc* a

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan

University of Technology, Wuhan, 430070, China b

Brigham and Women’s Hospital, Harvard Medical School, Boston, 02115, USA

c

School of Engineering and Applied Sciences, Harvard University, Cambridge, 02138, USA

d

Department of Orthopedics, Second Hospital of Yueyang, Yueyang, 414000, China

e

College of Environment and Resources, Fuzhou University, Fuzhou 350108, China.

f

School of Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430205,

China g

Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK

KEYWORDS: Nerve regeneration; Nerve growth factor; Nerve injury; Hypoxia; Tacrolimus

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ABSTRACT

Treatment of injured peripheral nerve, especially the long-distant nerve defect remains a significant challenge in regenerative medicine due to complex biological conditions and lack of biomaterials for effective nerve reconstruction. Without proper treatment, the injury to nerve leads to motor and sensory dysfunction. Here, we have developed an efficacious nerve allograft treated with dual drug containing Tacrolimus (FK506) and nerve growth factor (NGF) to bridge the nerve gap and achieve rapid neural tissue recovery without immunological rejection. The nerve recovery of structure, activity, and function of rats treated with the dual drug treated allograft is investigated by walking track analysis and electrophysiological measurement. The sciatic functional index is measured to be -3.0 after 12-week treatment. The nerve conduction velocity, peak latency, and peak amplitude of the nerve action potentials demonstrate the nerve functional recovery. To study the synergistic effect of dual drug on the growth of neuritis, a neural cell hypoxic model was created. The dual drug exhibited the high efficiency to promote the growth of nerve cells under the nerve injury-induced hypoxic condition. The dual drug could provide protection of the cells against anti-oxidative damage from hypoxia by the expression of heat shock protein, hypoxia-inducible factor, β-tubulin, and vimentin. e principal findings, and point out major conclusions.


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Introduction

Approximately 200,000 cases of peripheral nerve injury treatment are performed annually in the United States.1 In Europe, 300,000 treatments are carried out annually to repair injured peripheral nerves.2 These injuries are usually accompanied by ischemic and hypoxic nerve damages.3-5 The current clinical practice to repair the peripheral nerve injury involves nerve autografts, in which autologous tissues replace injured peripheral nerves.6 In addition to its low immunological reactions, the nerve autografts also provide biocompatible microenvironments to promote therapeutic effects.7 However, low patient compliance due to multiple surgeries and donor site complications limit further development of nerve autograft in clinical practice.8-9 In the last decade, processed or acellular nerve allografts have been employed in the clinic to replace nerve autografts, since they provide a highly accurate donor, and do not sacrifice expendable donor nerve that is associated with multiple surgeries, loss of senses and increased risk of the neuroma.10 A critical challenge of allografts is the immunorejection of the patient.11 Tacrolimus (FK506) is a FDA-approved immunosuppressant in allografts due to its inhibition of phosphorylation of nitric oxide synthase (NOS) to its catalytic activity.12 Thus, FK506 can effectively reduce NO formation, which prevents the neurotoxicity.13 In addition to its function in immunosuppression, FK506 can promote neurotrophic efficacy, where axonal re-growth can be accelerated with recovered functions after the surgical treatment of peripheral nerve and spinal cord injury.14-18 For example, FK506 at a low dose of 0.1 mg kg-1 can assist the recovery of nerve morphology and functions, where the outcome of the treatment is identical to an untreated nerve.19 Nerve growth factor (NGF) serves as an essential component in the regulation of peripheral and central nerve regeneration, growth differentiation and functions. The mediation of NGF in inducing cell differentiation and viability is achieved by signaling pathways including


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Ras/Raf/MAP kinase c-N-terminal, and phospholipase C.20 The limitations of sole NGF in therapeutic application are the low yield and delivery efficiency to target sites, and insufficient action time for regeneration.21 Since FK506 can be readily produced and penetrate through the blood-brain barrier, synergic effect of FK506 and NGF may be of use in promoting neuronal repair. Here, nerve allografts treated with dual drug comprised of FK506 and NGF were developed to treat peripheral nerve injury. The synergistic effect of FK506 and NGF within the allograft on nerve regeneration was assessed in an animal model. Walking track analysis was used to evaluate the recovery of the injured nerve of the animal implanted with FK506/NGF treated

allograft.

Functional

reconstruct

of

the

injured

nerve

was

measured

by

electrophysiological tests. Structure recovery of regenerated sciatic nerve postoperatively was measured by immunohistological staining and thickness characterization of the myelin sheath. To further understanding of the mechanism of the FK506/NGF dual drug promoting nerve regeneration, the hypoxic effect of nerve cells treated with FK506/NGF were studied. Nerve cell differentiation was assessed by measuring neurite length elongation. The expression levels of hypoxia-inducible factor (HIF-1α), hot shock protein (HSP70), axolemma protein (GAP-43), cell skeleton protein (β-tubulin) and vimentin protein in rats with peripheral nerve injury were measured by Western-blot methods.

Results and Discussion Decellularized allograft was implanted in a rat with left sciatic nerve gap (10 mm segment), which was used as a nerve injured model (Figure 1a-i, b). The proximal and the distal stumps were connected by the allograft using 9-0 nylon sutures (Figure 1b inset). Dual drugs comprising FK506 at a dose of (1 mg kg-1) and NGF (20 ng kg-1) were injected subcutaneously once a day


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for a week (Figure 1a-ii).26-28 The formation of the bundles of axons (Figure 1a-iii) and the proliferation of Schwann cells (Figure 1a-iv) were evaluated for nerve regeneration. The nerve regeneration was proved by post-operation after 1 month (Figure 1b). The FK506/NGF treated allograft was measured to be 1.1 fold thicker than sole NGF and sole FK506 treated allografts, and 1.3 fold thicker than pure allograft (Figure 1c). Without any drug treatment, a swollen and tangled mass was observed in the allograft that might be resulted from connective tissue fibrosis. The allograft with low uniformity could lead to painful neuroma formation that may affect nerve functional recovery.29 The uniformity of allograft was significantly improved 6.4 fold by functionalizing with sole NGF and 1.4 fold with sole FK506 that functions in immunesuppression which could inhibit connective tissue fibrosis (Figure 1c inset, Figure S1).30 The uniformity of allograft treated with FK506/NGF was improved 11 fold than untreated counterpart, demonstrating that the synergic effect of FK506 and NGF could improve allograft formation and nerve functional recovery. The nerve functional recovery was further evaluated by triceps wet weight after 1 month post-operation (Figure 1d). A significant increase in the recovery rate of muscle wet weight was observed in FK506/NGF treated muscle. The measured sciatic nerve recovery rate of FK506/NGF treated group was 1.4 fold higher than sole FK506, and 1.6 fold higher than sole NGF and untreated counterparts. The motor and functional recovery by the regenerative nerve was evaluated by a standard walking track analysis after postoperative 1, 4, and 12 weeks. A rat implanted with an allograft was confined in a walking pathway, where a white sheet was placed at the bottom. The rat footprints were recorded on the sheet by staining rat feet with blue ink, where the photographs were captured by a digital camera for analysis. The nerve functional recovery from nerve lesion was evaluated by SFI,23 which was calculated from EPL, ETS and EIT of footprints (Eq. S1 and S2). The SFI of a normal rat footprint was measured


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at 0 ± 5.3 (Figure 1e-i). As compared with a rat implanted with pure allograft (control), the SFI value of sole FK506 treated rat decreased 2.6% after postoperative 4 weeks. Although no significant difference was observed with/out NGF treatment, the synergistic effect of FK506 and NGF increased the SFI value to 6.6%. After 12 weeks post-operation, the SFI value of FK506/NGF treated rat was -3.0, which was nearly 3 fold higher than all other counterparts (Figure 1e-ii, iii). Moreover, no obvious significant differences were observed in the morphology of the recovered allograft, the recovery rate of triceps and footprint after 12 weeks between FK506/NGF-treated rats and normal rats, which indicated that the synergistic effect of FK506 and NGF could improve nerve regeneration from injury.


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Figure 1. Evaluation of FK506/NGF treated allograft in vivo. (a) Schematic of FK506/NGF treated allograft. (i) Decellularized allograft; (ii) Dual drug treatment on the allograft; (iii) polarized vessel formation; (iv) Schwann cells migration along the vasculature from axons to targets (nerve regeneration). (b) Implantation of a FK506/NGF treated allograft nerve before and after suture (inset). Scale bar=5 mm. Quantitative evaluation of FK506/NGF treated allograft and triceps 1 month postoperation: (c) comparison of allograft thickness and uniformity distribution


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(inset) among control samples (pure allografts), NGF-treated allografts, FK506-treated allografts, FK506/NGF-treated allografts, and normal samples. (d) Comparison of recovery rate of triceps wet weight at the post-operation. Inset images show the structures of postoperation triceps. Scale bar=10 mm. (e) Walking track analysis of rat foot. (i) Photographs of a normal rat footprint. Scale bar=3 mm. (NPL) Normal print length, (NTS) normal toe spread, (NIT) normal intermediary toe spread; (ii) Footprint photographs and (iii) quantitative measurements of rats implanted with pure allografts, sole NGF, sole FK506 and FK506/NGF-treated allografts after postoperative 1, 4 and 12 weeks, respectively. n=5. Scale bar = 5 mm. **: Compared to other groups, p < 0.01. The nerve conduction recovery after implantation was evaluated by using electrophysiological tests. Rats after postoperative 12 weeks were anesthetized, and the operated surgical sites were re-opened to expose the sciatic nerve. A stimulating electrode was inserted into the proximal neural stem of the regenerated nerve, and the recording electrode was inserted into the calf triceps (Figure 2a). Quantitative measurement of nerve functional recovery was evaluated by nerve conduction velocity (NCV), peak latency (LAT) and peak amplitude (AMP) of the sciatic nerve action potentials (Figure 2b-d, Figure S2). Implanted NGF treated and FK506 treated allograft promoted 29% and 20% NCV as compared to the pure allograft group (control) (Figure 2b). Cooperating FK506 with NGF, NCV could be improved by 17%, indicating that FK506/NGF may accelerate recovery of nerve electrical activity. In addition, FK506/NGF group showed 37% lower LAT than other counterparts (NGF treated allograft, FK506 treated allograft, pure allograft), demonstrating that FK506/NGF could shorten the time delay between the electrical stimulus and the reaction time (Figure 2c). Moreover, higher voltage difference was detected in allograft treated with FK506/NGF as compared to sole NGF allograft, sole FK506


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allograft and pure allograft groups (Figure 2d). The improved electrophysiological activity of FK506/NGF treated groups as compared to sole NGF, sole FK506 and control groups was consistent with the recovery rate of triceps wet weight measurement (Figure 1d). Hence, it was hypothesized that the dual drug could improve nerve function recovery.

Figure 2. Effect of dual drug on the function of regenerated sciatic nerve postoperatively. (a) (i) Scheme of nerve conduction study assessed in a rat model. Photographs show the operation of the electrophysiological tests in (ii) low and (iii) high magnifications. Scale bar=10 cm. (b) Latency of the sciatic nerve 12 weeks postoperatively. (c) Amplitude of the sciatic nerve 12 weeks postoperatively. (d) Nerve conduction velocity of the sciatic nerve 12 weeks


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postoperatively. Compared to other groups, *p < 0.05, **p

Tacrolimus and Nerve Growth factor Treated Allografts for Neural

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