Neuroscience Letters 566 (2014) 177–181
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Local delivery of controlled released nerve growth factor promotes sciatic nerve regeneration after crush injury Hailong Yu 1 , Jun Liu 1 , Junxiong Ma, Liangbi Xiang ∗ Department of Orthopedics, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang, 110016 Liaoning, China
h i g h l i g h t s • The beneficial effect of controlled released NGF-microspheres on nerve regeneration after crush injury was investigated. • This NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration after crush injury. • NGF-microspheres have the potential to be a neuroprotective agent for nerve crush injury repair applications.
a r t i c l e
i n f o
Article history: Received 21 December 2013 Received in revised form 24 February 2014 Accepted 26 February 2014 Keywords: Nerve growth factor (NGF) Microsphere Controlled released Nerve crush injury Peripheral nerve
a b s t r a c t Controlled released nerve growth factor (NGF)-microspheres are able to enhance peripheral nerve regeneration across short nerve gaps. However, such beneficial effect has never been investigated in crush injury model in vivo. The present study was designed to investigate such a possibility. The rats subjected to sciatic nerve crush injury were intraperitoneally administrated daily for 4 weeks with NGF or normal saline or locally injected controlled released NGF-microspheres only once. Sham operation group without injury was defined as normal group. Nerve regeneration was investigated by morphometric analysis. The recovery of nerve function was estimated by electrophysiological analysis, behavioral tests and morphometric observation of the denervated muscles. The results showed that both NGF and NGF-microspheres improved nerve regeneration and recovery of nerve function. In addition, NGF-microspheres achieved better results than NGF group. These findings show that this controlled released NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration and recovery of motor and sensory function after sciatic nerve crush injury in rats, indicating that the NGF-microspheres have the potential to be a neuroprotective agent for nerve crush injury repair applications. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peripheral nerve crush injury results in serious problems, such as restricted activity and life-long disability [1]. Although medical science have been improved over the past decades, the outcome of nerve crush injury is unsatisfactory. As the continuity of the nerve was still intact, microsurgery provided little help. Drug therapy was the major way to promote the nerve regeneration [2]. Therefore, increasing the curative effect of existing drugs, such as neurotrophic factors, has gained extensive attention. Neurotrophic factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), improve the regeneration of injured nerves [3–9]. NGF, which was broadly used for nerve injury, stimulates the growth of nerve fiber and dictate the direc-
∗ Corresponding author. Tel.: +86 024 28856247; fax: +86 024 28856247. E-mail addresses: [email protected], [email protected] (L. Xiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.02.065 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
tion of nerve regeneration [10]. However, the half life period of NGF was so short that it is quickly inactivated under physiological condition [11], which may limit its curative effect. To maintain the bioactivity of NGF, many kinds of control-released NGF have been developed in the past decade. But the stability and duration of the control-released systems were still unsatisfactory. Therefore, a kind of double-controlled released system of NGF was developed in our previous study, which consists of microspheres of NGF fixed by fibrin glue [12]. The NGF was control-released both by microsphere and fibrin glue, which could significantly prolong the release of NGF. The results showed that it was capable of enhancing peripheral nerve regeneration across short nerve gaps repaired with acellular nerve grafts, suggesting the possibility of developing this control release system a neuroprotective drug for peripheral nerve repair applications. But such a beneficial effect has never been investigated in crush injury model in vivo. Therefore, the present study was designed to investigate the beneficial effect of the
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H. Yu et al. / Neuroscience Letters 566 (2014) 177–181
controlled-released NGF-microspheres on nerve regeneration and functional recovery after crush injury of sciatic nerve in rats.
plate was removed to avoid burn, and the TWRL was considered as 12 s.
2. Materials and methods 2.5. Electrophysiological test 2.1. Surgical procedures and drug treatments All animal procedures were performed under a protocol reviewed and approved by the Institutional Ethical Committee of General Hospital of Shenyang Military Area Command of Chinese PLA. Young adult male Sprague-Dawley rats (n = 48, provided by the Laboratory Animal Center of the General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang, China), weighing from 200 g to 240 g, were anesthetized (40 mg/kg body weight) by intraperitoneal injection of sodium pentobarbital solution (10 mg/ml). The left sciatic nerves in all rats were exposed. Twelve rats (n = 12) were randomly selected as normal group (sham operation group without injury), and the skin was then closed with 4-0 stitches. The left sciatic nerves of the other rats were crushed at the site 5 mm proximal to the bifurcation using a pair of forceps for 3 times (10 s each time) with an interval of 10 s, and the skin was then closed with 4-0 stitches. Following surgery, all rats were randomly divided into three groups (NGF-microsphere group, NGF group, control group; n = 12 in each group). For the NGF-microsphere group, 1 ml fibrin glue with 10 mg microencapsulated NGF was injected around the injury site. 80 ng NGF/day for the NGF group, and normal saline for both the control group and normal group were intraperitoneally administrated daily for 4 weeks, respectively. The dosages of NGF for both group was decided according to our previous study and preliminary experiment [12]. 2.2. Behavioral analysis To evaluate the motor functional recovery, walking track analysis was performed on all animals every week following surgery [13]. The changes of paw prints were recorded to calculate sciatic functional index (SFI) follows the one proposed by Bain et al. [13]: SFI =
−38.3 ×
+
13.3 ×
EPL + NPL NPL EIT + NIT NIT
+ 109.5 ×
ETS + NTS NTS
− 8.8
Electrophysiological test was performed every week after the surgery according to the previous study [15]. The recording electrode was placed in the gastrocnemius muscle to record compound muscle action potentials (CMAPs). CMAPs of unoperated side were measured as normal CMAPs. The CMAP peak amplitude, CMAP latency of onset, and nerve conduction velocity (NCV) values were calculated [16].
2.6. Morphometric analysis Four weeks after the surgery, the sciatic nerves were harvested for morphometric analysis. The distal portion of the nerves was prepared into 1.0 m semi-thin sections and 50 nm ultra-thin sections. The 1.0 m semi-thin sections were stained with toluidine blue/borax solution, and were observed under a light microscope (AH3, Olympus, Tokyo, Japan). 50 nm ultra-thin sections were stained with uranyl acetate/lead citrate, and were observed under a transmission electron microscope (H-600, HITACHI, Tokyo, Japan). For each sample, three randomly chosen fields were selected for measurement of the total nerve area, the number of medullated nerve fibers, and the diameter of axon and fiber. The nerve regeneration was assessed by the total number of myelinated axons per nerve transverse section and the mean diameter of nerve fibers [15]. The myelination was estimated by G-ratio (the axon to fiber diameter ratio, G-ratio = axon diameter/fiber diameter). Four weeks after surgery, the gastrocnemius muscles in the operated limbs were harvested, and fixed with formalin. The samples were embedded with paraffin and stained by Masson trichrome staining. Then the samples were observed under a light microscope. For each sample, three randomly chosen fields were selected for measurement of the total area and the muscle fiber area, and the percentage of the latter to the former was calculated [15]. All the morphometric analysis were blind conducted by examiners.
2.7. Statistical analysis 2.3. Mechanical withdrawal thresholds To evaluate the sensory functional recovery, paw withdrawal threshold in response to a mechanical stimulus was measured 4 weeks after the treatment using a series of von Frey filaments [14]. All the animals were placed on a mental mesh floor. The von Frey filaments were applied to the mid-plantar surface of the operated hind paw for 6–8 s. Filaments were applied in ascending order, which ranged from 47 mN to 156 mN. The smallest force which elicits foot withdrawal response was recorded as the mechanical withdrawal threshold.
All data are expressed as the mean ± standard error of mean (S.E.M). One-way or two-way analysis of variance (ANOVA) was used to compare mean values with the SPSS13.0 software package (SPSS Inc., Chicago, IL, USA). Tukey post hoc test was used to make comparisons. Values of p < 0.05 were considered statistically significant.
3. Results
2.4. Hot plate test
3.1. NGF-microsphere promotes nerve regeneration after sciatic nerve crush injury
To evaluate the sensory functional recovery, hot plate test was performed on all the animals 4 weeks after the treatment following a previous study [1]. Briefly, a hot plate at 56 ◦ C was prepared for the test. The animal was positioned to stand with the operated hind paw on the hot plate. The duration from the onset of paw-plate contact to hind paw withdraw was recorded as thermal withdrawal reflex (TWRL). All the test were repeated for 4 times, with a 5 min interval. If no hind paw withdraw was observed after 12 s, the hot
Four weeks after injury, the number of nerve fibers and the mean fiber diameter in NGF-microsphere group and NGF group were significantly higher than that in control group (p < 0.05; Table 1 and Fig. 1). G-ratio (as an indicator of myelination) in NGF-microsphere group and NGF group were significantly better than that in control group (p < 0.05; Table 1 and Fig. 1). In addition, the morphometric indices in NGF-microsphere group were significantly better than those in NGF groups (p < 0.05; Table 1 and Fig. 1).
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Table 1 Histological assessment (n = 12, mean ± SEM). Group
Fiber counts (fb/mm2 )
Normal group Control group NGF group NGF-microsphere group
26,212 20,012 23,768 25,156
± ± ± ±
Fiber diameter (m)
3011 1233* 2986*,** 3032*,**,***
4.21 2.87 3.85 4.05
± ± ± ±
0.44 0.12* 0.32*,** 0.39*,**,***
G-ratio (axon to fiber diameter ratio) 0.604 0.812 0.712 0.652
± ± ± ±
0.106 0.164* 0.122*,** 0.114*,**,***
The total nerve area, the number of medullated nerve fibers, the mean diameter of the axon and the fiber was measured. The G-ratio was calculated. More fiber counts and bigger diameter of the fiber might purport better nerve regeneration, and the lower G-ratio might purport thicker medulla sheath and better myelination. * Compared with Normal group, p < 0.05. ** Compared with Control group, p < 0.05. *** Compared with NGF group, p < 0.05. Table 2 The sciatic functional index (SFI) values at predetermined time postoperatively (n = 12). Weeks after surgery
Normal group
Control group
NGF group
NGF-microsphere group
1 2 3 4
−28.22 −28.15 −28.25 −28.14
−93.32 −83.89 −68.98 −46.27
−94.95 −78.95 −58.12 −35.27
−93.22 −73.88 −50.30 −30.25
* ** ***
± ± ± ±
2.65 2.57 2.61 2.53
± ± ± ±
5.59* 4.68* 4.94* 3.15*
± ± ± ±
5.55* 4.88*,** 4.68*,** 3.22*,**
± ± ± ±
5.50* 4.82*,**,*** 4.27*,**,*** 2.43*,**,***
Compared with Normal group, p < 0.05. Compared with Control group, p < 0.05. Compared with NGF group, p < 0.05.
3.2. NGF-microsphere promotes recovery of motor function after sciatic nerve crush injury One week after injury, the SFI values in the control group, NGF group and NGF-microsphere group were in the similar range (p > 0.05, Table 2). Two to four weeks after injury, the SFI values in NGF group and NGF-microsphere group were significantly higher than that in control group (p < 0.05, Table 2). Furthermore, the SFI values in NGF-microsphere group were significantly higher than that in NGF group at 2–4 weeks (p < 0.05, Table 2), indicating that NGF-microsphere might achieve better recovery of motor function than routinely administrated-NGF after crush injury in rats. At the 4 week end point, the CMAP peak amplitude, CMAP latency of onset, and NCV values in NGF group and NGFmicrosphere group were significantly better than that in control group (p < 0.05, Table 3). In addition, all the electrophysiological values in NGF-microsphere group were significantly better than those in the NGF groups (p < 0.05, Table 3).
Four weeks after treatment, atrophy of gastrocnemius muscle was observed in control group. However, the atrophy was significantly reversed by application of NGF. The average percentage of muscle fiber area of gastrocnemius muscle in NGF group and NGFmicrosphere group were significantly better than that in control group (p < 0.05, Fig. 2). In addition, the average percentage of muscle fiber area in NGF-microsphere group was significantly better than that in NGF groups (p < 0.05, Fig. 2). 3.3. NGF-microsphere promotes recovery of sensory function after sciatic nerve crush injuries Four weeks after injury, the withdrawal threshold in NGF group and NGF-microsphere group were significantly lower than that in control group (p < 0.05, Fig. 3), indicating recovery of sensory function in NGF group and NGF-microsphere group. Furthermore, the withdrawal threshold in NGF-microsphere group were significantly lower than that in NGF group (p < 0.05, Fig. 3), indicating
Fig. 1. Representative images of transverse sectioned injured nerve under transmission electron microscope or light microscope in the Normal group (A and E), Control group (B and F), NGF group (C and G) and NGF-microsphere group (D and H) at 4 weeks after treatment. The mean diameter of the axon and the fiber was measured (i.e., the red line in A and C), and the G-ratio was calculated. Bigger diameter of the fiber might purport better nerve regeneration, and the lower G-ratio might purport thicker medulla sheath and better myelination. In A and D, the nerve fibers were in good order, the mean fiber diameter was bigger, the G-ratio was lower. The total number of myelinated axons per nerve transverse section was calculated. In E and H, the nerve fibers were in good order, and the total number of myelinated axons was larger, which might purport better nerve regeneration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 3 The electrophysiological values at predetermined time postoperatively (n = 12).
Nerve conduction velocity (m/s) Peak amplitude of CMAP (mV) Latency of CMAP onset (ms) * ** ***
Normal group
Control group
NGF group
NGF-microsphere group
35.22 ± 3.21 41.22 ± 4.06 0.79 ± 0.072
17.23 ± 1.35* 16.12 ± 1.42* 2.15 ± 0.146*
25.87 ± 2.73*,** 24.3 ± 2.65*,** 1.72 ± 0.182*,**
33.92 ± 3.04*,**,*** 39.42 ± 3.76*,**,*** 0.88 ± 0.078*,**,***
Compared with Normal group, p < 0.05. Compared with Control group, p < 0.05. Compared with NGF group, p < 0.05.
that NGF-microsphere might achieve better recovery of sensory function than routinely administrated-NGF after crush injury in rats. Function of thermal nociceptive sensation was assessed by the hot plate test. Four weeks after injury, the TWRL values in NGF group and NGF-microsphere group were significantly lower than that in control group (p < 0.05, Fig. 3), indicating recovery of sensory function in NGF group and NGF-microsphere group. Furthermore, the TWRL values in NGF-microsphere group were significantly
lower than that in NGF group (p < 0.05, Fig. 3), indicating that NGFmicrosphere might achieve better recovery of sensory function than routinely administrated-NGF after crush injury in rats. 4. Discussion The present study investigated the possibility that local injection of controlled released NGF-microspheres might be capable of enhancing nerve regeneration and promoting functional
Fig. 2. Representative images of gastrocnemius muscle by H–E staining in the Normal group (A), Control group (B), NGF group (C), and NGF-microsphere group (D) at 4 weeks after treatment. The sum of muscle fiber cross-sectional areas in a field divided by the total area of the field multiplied by 100 (E) were shown. *Compared with Normal group, p < 0.05. # Compared with Control group, p < 0.05. ˆ Compared with NGF group, p < 0.05.
Fig. 3. Statistical analysis of mechanical withdraw threshold (A) and thermal withdraw threshold (B). *Compared with Normal group, p < 0.05; # Compared with Control group, p < 0.05; ˆ Compared with NGF group, p < 0.05.
H. Yu et al. / Neuroscience Letters 566 (2014) 177–181
recovery after sciatic nerve crush injury in rats. We found that NGF-microsphere was capable of promoting nerve regeneration and accelerating recovery of motor and sensory function after crush nerve injury. Further studies showed that local injection of controlled released NGF-microspheres achieved better nerve regeneration than intraperitoneally daily-administration of NGF. All the findings raise the possibility that controlled released NGFmicrospheres might enhance the curative effect of NGF, and might be a better neuroprotective agent for nerve crush injury. The therapy of nerve injury varied with the type of injury. For nerve section, microsurgery was generally performed for architectural reconstruction of the nerve, while drug therapy was used to promote the nerve regeneration. However, for nerve crush injury, as the continuity of the nerve was still intact, microsurgery provided little help. Drug therapy was the major therapeutic tool to promote the nerve regeneration after nerve crush injury. Although the medical science has been improved in the past decades, the outcome of nerve crush injury is unsatisfactory. As drug therapy was the major therapeutic tool to promote the nerve regeneration after nerve crush injury, increasing the curative effect of existing drugs, such as neurotrophic factors, may have more senses. NGF, as a commonly used neuroprotective agent, plays a critical role in nerve repair after injury. Previous studies proved that NGF was important for differentiation and survival of sensory and sympathetic neurons [5,17], and was cable of inducing nerve regeneration [10]. However, due to the rapid diffusion and deactivation in extracellular fluids, it is difficult for NGF to be retained at the injury sites, which limits the curative effect. In order to maintain the local concentration of NGF, a kind of controlled released NGF-microspheres was developed in our previous study [12], which was proved to be capable of enhancing peripheral nerve regeneration across short nerve gaps repaired with acellular nerve grafts. In the present study, morphometric analysis showed better nerve recovery in NGF group, and the best recovery in NGF-microsphere group. All those findings, together with the findings in the present study, highlight the beneficial effect of controlled released NGF-microspheres on nerve regeneration after nerve crush injury. Electrophysiology analysis was used to investigate the recovery of motor function after local injection of NGF-microspheres. As the CAMP amplitude is closely related to the number of nerve fibers innervating the muscle, it is possible to further evaluate the recovery of motor function [16]. In this study, better CAMP latency, NCV and CAMP peak amplitude were observed in both NGF group and NGF-microsphere group than those in the control group, indicating better recovery of motor function after NGF or NGF-microsphere treatment. Interestingly, the electrophysiological results in NGFmicrosphere group were better than those in NGF group, indicating that this controlled released system of NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration. In addition, the atrophy of the denervated muscles was partially reversed in NGF group and mostly reversed in NGF-microsphere group. The behavioral appearances were partially improved in NGF group and significantly improved in NGF-microsphere group. All these findings indicated that NGF-microspheres could enhance the beneficial effect of NGF on recovery of motor function. Further studies showed that NGF-microspheres and NGF could also improve the recovery of thermal and mechanical sensory function. Furthermore, NGFmicrospheres might result in a better recovery than NGF, indicating that NGF-microspheres could enhance the beneficial effect of NGF on recovery of sensory function. In conclusion, this controlled released NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration and recovery of motor and sensory function after nerve crush injury in
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rats, indicating that NGF-microspheres have the potential to be a neuroprotective agent for nerve injury repair applications for nerve crush injury. Further study should be done to confirm those effects in larger animals or even humans before clinical application. Competing interests The authors have declared that no competing interest exists. Acknowledgements This research was supported by the Science and Key Technology Research and Development Program of Liaoning Province (No. 2011225041 and No. 2012225019), and “Twelfth Five-Year Plan” Scientific Research Funds Project of Chinese PLA (CWS11J209). References [1] J. Noble, C.A. Munro, V.S. Prasad, R. Midha, Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries, J. Trauma 45 (July (1)) (1998) 116–122, PMID: 9680023. [2] M. Schumacher, R. Guennoun, D.G. Stein, A.F. De Nicola, Progesterone: therapeutic opportunities for neuroprotection and myelin repair, Pharmacol. Ther. 116 (October (1)) (2007) 77–106, PMID: 17659348. [3] W. Marcol, K. Kotulska, M. Larysz-Brysz, I. Matuszek, E. Olakowska, J. LewinKowalik, Extracts obtained from predegenerated nerves improve functional recovery after sciatic nerve transection, Microsurgery 25 (6) (2005) 486–494, PMID: 16134094. [4] D.J. Bryan, A.H. Holway, K.K. Wang, A.E. Silva, D.J. Trantolo, D. Wise, I.C. Summerhayes, Influence of glial growth factor and Schwann cells in a bioresorbable guidance channel on peripheral nerve regeneration, Tissue Eng. 6 (April (2)) (2000) 129–138, PMID: 10941208. [5] A.I. Gravvanis, D.A. Tsoutsos, G.A. Tagaris, A.E. Papalois, C.G. Patralexis, T.G. Iconomou, P.N. Panayotou, J.D. Ioannovich, Beneficial effect of nerve growth factor-7S on peripheral nerve regeneration through inside-out vein grafts: an experimental study, Microsurgery 24 (5) (2004) 408–415, PMID: 15378588. [6] E.G. Fine, I. Decosterd, M. Papaloïzos, A.D. Zurn, P. Aebischer, GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap, Eur. J. Neurosci. 15 (February (4)) (2002) 589–601, PMID: 11886440. [7] B. Hontanilla, C. Aubá, O. Gorría, Nerve regeneration through nerve autografts after local administration of brain-derived neurotrophic factor with osmotic pumps, Neurosurgery 61 (December (6)) (2007) 1268–1274, PMID: 18162907 (discussion 1274–1275). [8] X. Santos, J. Rodrigo, B. Hontanilla, G. Bilbao, Local administration of neurotrophic growth factor in subcutaneous silicon chambers enhances the regeneration of the sensory component of the rat sciatic nerve, Microsurgery 19 (6) (1999) 275–280, PMID: 10469442. [9] R. Ikeguchi, R. Kakinoki, T. Matsumoto, T. Yamakawa, K. Nakayama, Y. Morimoto, H. Tsuji, J. Ishikawa, T. Nakamura, Basic fibroblast growth factor promotes nerve regeneration in a C− -ion-implanted silicon chamber, Brain Res. 1090 (May (1)) (2006) 51–57, PMID: 16677621. [10] S.D. Skaper, Nerve growth factor: a neurokine orchestrating neuroimmuneendocrine functions, Mol. Neurobiol. 24 (August–December (1–3)) (2001) 183–199, PMID: 11831552. [11] C.C. Tsai, M.C. Lu, Y.S. Chen, C.H. Wu, C.C. Lin, Locally administered nerve growth factor suppresses ginsenoside Rb1-enhanced peripheral nerve regeneration, Am. J. Chin. Med. 31 (5) (2003) 665–673, PMID: 14696670. [12] H. Yu, J. Peng, Q. Guo, L. Zhang, Z. Li, B. Zhao, X. Sui, Y. Wang, W. Xu, S. Lu, Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor, Microsurgery 29 (4) (2009) 330–336, PMID: 19296502. [13] J.R. Bain, S.E. Mackinnon, D.A. Hunter, Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat, Plast. Reconstr. Surg. 83 (January (1)) (1989) 129–138, PMID: 2909054. [14] C.F. Vogelaar, D.H. Vrinten, M.F. Hoekman, J.H. Brakkee, J.P. Burbach, F.P. Hamers, Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome, Brain Res. 1027 (November (1–2)) (2004) 67–72, PMID: 15494158. [15] J. Ma, W. Li, R. Tian, W. Lei, Ginsenoside Rg1 promotes peripheral nerve regeneration in rat model of nerve crush injury, Neurosci. Lett. 478 (2) (2010) 66–71, PubMed: 20438804. [16] X. Hu, J. Huang, Z. Ye, L. Xia, M. Li, B. Lv, X. Shen, Z. Luo, A novel scaffold with longitudinally oriented microchannels promotes peripheral nerve regeneration, Tissue Eng. A 15 (November (11)) (2009) 3297–3308, PMID: 19382873. [17] L. Aloe, Rita Levi-Montalcini: the discovery of nerve growth factor and modern neurobiology, Trends Cell Biol. 14 (July (7)) (2004) 395–399, PMID: 15246433.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Local delivery of controlled released nerve growth factor promotes sciatic nerve regeneration after crush injury Hailong Yu 1 , Jun Liu 1 , Junxiong Ma, Liangbi Xiang ∗ Department of Orthopedics, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang, 110016 Liaoning, China
h i g h l i g h t s • The beneficial effect of controlled released NGF-microspheres on nerve regeneration after crush injury was investigated. • This NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration after crush injury. • NGF-microspheres have the potential to be a neuroprotective agent for nerve crush injury repair applications.
a r t i c l e
i n f o
Article history: Received 21 December 2013 Received in revised form 24 February 2014 Accepted 26 February 2014 Keywords: Nerve growth factor (NGF) Microsphere Controlled released Nerve crush injury Peripheral nerve
a b s t r a c t Controlled released nerve growth factor (NGF)-microspheres are able to enhance peripheral nerve regeneration across short nerve gaps. However, such beneficial effect has never been investigated in crush injury model in vivo. The present study was designed to investigate such a possibility. The rats subjected to sciatic nerve crush injury were intraperitoneally administrated daily for 4 weeks with NGF or normal saline or locally injected controlled released NGF-microspheres only once. Sham operation group without injury was defined as normal group. Nerve regeneration was investigated by morphometric analysis. The recovery of nerve function was estimated by electrophysiological analysis, behavioral tests and morphometric observation of the denervated muscles. The results showed that both NGF and NGF-microspheres improved nerve regeneration and recovery of nerve function. In addition, NGF-microspheres achieved better results than NGF group. These findings show that this controlled released NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration and recovery of motor and sensory function after sciatic nerve crush injury in rats, indicating that the NGF-microspheres have the potential to be a neuroprotective agent for nerve crush injury repair applications. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peripheral nerve crush injury results in serious problems, such as restricted activity and life-long disability [1]. Although medical science have been improved over the past decades, the outcome of nerve crush injury is unsatisfactory. As the continuity of the nerve was still intact, microsurgery provided little help. Drug therapy was the major way to promote the nerve regeneration [2]. Therefore, increasing the curative effect of existing drugs, such as neurotrophic factors, has gained extensive attention. Neurotrophic factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), improve the regeneration of injured nerves [3–9]. NGF, which was broadly used for nerve injury, stimulates the growth of nerve fiber and dictate the direc-
∗ Corresponding author. Tel.: +86 024 28856247; fax: +86 024 28856247. E-mail addresses: [email protected], [email protected] (L. Xiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.02.065 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
tion of nerve regeneration [10]. However, the half life period of NGF was so short that it is quickly inactivated under physiological condition [11], which may limit its curative effect. To maintain the bioactivity of NGF, many kinds of control-released NGF have been developed in the past decade. But the stability and duration of the control-released systems were still unsatisfactory. Therefore, a kind of double-controlled released system of NGF was developed in our previous study, which consists of microspheres of NGF fixed by fibrin glue [12]. The NGF was control-released both by microsphere and fibrin glue, which could significantly prolong the release of NGF. The results showed that it was capable of enhancing peripheral nerve regeneration across short nerve gaps repaired with acellular nerve grafts, suggesting the possibility of developing this control release system a neuroprotective drug for peripheral nerve repair applications. But such a beneficial effect has never been investigated in crush injury model in vivo. Therefore, the present study was designed to investigate the beneficial effect of the
178
H. Yu et al. / Neuroscience Letters 566 (2014) 177–181
controlled-released NGF-microspheres on nerve regeneration and functional recovery after crush injury of sciatic nerve in rats.
plate was removed to avoid burn, and the TWRL was considered as 12 s.
2. Materials and methods 2.5. Electrophysiological test 2.1. Surgical procedures and drug treatments All animal procedures were performed under a protocol reviewed and approved by the Institutional Ethical Committee of General Hospital of Shenyang Military Area Command of Chinese PLA. Young adult male Sprague-Dawley rats (n = 48, provided by the Laboratory Animal Center of the General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang, China), weighing from 200 g to 240 g, were anesthetized (40 mg/kg body weight) by intraperitoneal injection of sodium pentobarbital solution (10 mg/ml). The left sciatic nerves in all rats were exposed. Twelve rats (n = 12) were randomly selected as normal group (sham operation group without injury), and the skin was then closed with 4-0 stitches. The left sciatic nerves of the other rats were crushed at the site 5 mm proximal to the bifurcation using a pair of forceps for 3 times (10 s each time) with an interval of 10 s, and the skin was then closed with 4-0 stitches. Following surgery, all rats were randomly divided into three groups (NGF-microsphere group, NGF group, control group; n = 12 in each group). For the NGF-microsphere group, 1 ml fibrin glue with 10 mg microencapsulated NGF was injected around the injury site. 80 ng NGF/day for the NGF group, and normal saline for both the control group and normal group were intraperitoneally administrated daily for 4 weeks, respectively. The dosages of NGF for both group was decided according to our previous study and preliminary experiment [12]. 2.2. Behavioral analysis To evaluate the motor functional recovery, walking track analysis was performed on all animals every week following surgery [13]. The changes of paw prints were recorded to calculate sciatic functional index (SFI) follows the one proposed by Bain et al. [13]: SFI =
−38.3 ×
+
13.3 ×
EPL + NPL NPL EIT + NIT NIT
+ 109.5 ×
ETS + NTS NTS
− 8.8
Electrophysiological test was performed every week after the surgery according to the previous study [15]. The recording electrode was placed in the gastrocnemius muscle to record compound muscle action potentials (CMAPs). CMAPs of unoperated side were measured as normal CMAPs. The CMAP peak amplitude, CMAP latency of onset, and nerve conduction velocity (NCV) values were calculated [16].
2.6. Morphometric analysis Four weeks after the surgery, the sciatic nerves were harvested for morphometric analysis. The distal portion of the nerves was prepared into 1.0 m semi-thin sections and 50 nm ultra-thin sections. The 1.0 m semi-thin sections were stained with toluidine blue/borax solution, and were observed under a light microscope (AH3, Olympus, Tokyo, Japan). 50 nm ultra-thin sections were stained with uranyl acetate/lead citrate, and were observed under a transmission electron microscope (H-600, HITACHI, Tokyo, Japan). For each sample, three randomly chosen fields were selected for measurement of the total nerve area, the number of medullated nerve fibers, and the diameter of axon and fiber. The nerve regeneration was assessed by the total number of myelinated axons per nerve transverse section and the mean diameter of nerve fibers [15]. The myelination was estimated by G-ratio (the axon to fiber diameter ratio, G-ratio = axon diameter/fiber diameter). Four weeks after surgery, the gastrocnemius muscles in the operated limbs were harvested, and fixed with formalin. The samples were embedded with paraffin and stained by Masson trichrome staining. Then the samples were observed under a light microscope. For each sample, three randomly chosen fields were selected for measurement of the total area and the muscle fiber area, and the percentage of the latter to the former was calculated [15]. All the morphometric analysis were blind conducted by examiners.
2.7. Statistical analysis 2.3. Mechanical withdrawal thresholds To evaluate the sensory functional recovery, paw withdrawal threshold in response to a mechanical stimulus was measured 4 weeks after the treatment using a series of von Frey filaments [14]. All the animals were placed on a mental mesh floor. The von Frey filaments were applied to the mid-plantar surface of the operated hind paw for 6–8 s. Filaments were applied in ascending order, which ranged from 47 mN to 156 mN. The smallest force which elicits foot withdrawal response was recorded as the mechanical withdrawal threshold.
All data are expressed as the mean ± standard error of mean (S.E.M). One-way or two-way analysis of variance (ANOVA) was used to compare mean values with the SPSS13.0 software package (SPSS Inc., Chicago, IL, USA). Tukey post hoc test was used to make comparisons. Values of p < 0.05 were considered statistically significant.
3. Results
2.4. Hot plate test
3.1. NGF-microsphere promotes nerve regeneration after sciatic nerve crush injury
To evaluate the sensory functional recovery, hot plate test was performed on all the animals 4 weeks after the treatment following a previous study [1]. Briefly, a hot plate at 56 ◦ C was prepared for the test. The animal was positioned to stand with the operated hind paw on the hot plate. The duration from the onset of paw-plate contact to hind paw withdraw was recorded as thermal withdrawal reflex (TWRL). All the test were repeated for 4 times, with a 5 min interval. If no hind paw withdraw was observed after 12 s, the hot
Four weeks after injury, the number of nerve fibers and the mean fiber diameter in NGF-microsphere group and NGF group were significantly higher than that in control group (p < 0.05; Table 1 and Fig. 1). G-ratio (as an indicator of myelination) in NGF-microsphere group and NGF group were significantly better than that in control group (p < 0.05; Table 1 and Fig. 1). In addition, the morphometric indices in NGF-microsphere group were significantly better than those in NGF groups (p < 0.05; Table 1 and Fig. 1).
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Table 1 Histological assessment (n = 12, mean ± SEM). Group
Fiber counts (fb/mm2 )
Normal group Control group NGF group NGF-microsphere group
26,212 20,012 23,768 25,156
± ± ± ±
Fiber diameter (m)
3011 1233* 2986*,** 3032*,**,***
4.21 2.87 3.85 4.05
± ± ± ±
0.44 0.12* 0.32*,** 0.39*,**,***
G-ratio (axon to fiber diameter ratio) 0.604 0.812 0.712 0.652
± ± ± ±
0.106 0.164* 0.122*,** 0.114*,**,***
The total nerve area, the number of medullated nerve fibers, the mean diameter of the axon and the fiber was measured. The G-ratio was calculated. More fiber counts and bigger diameter of the fiber might purport better nerve regeneration, and the lower G-ratio might purport thicker medulla sheath and better myelination. * Compared with Normal group, p < 0.05. ** Compared with Control group, p < 0.05. *** Compared with NGF group, p < 0.05. Table 2 The sciatic functional index (SFI) values at predetermined time postoperatively (n = 12). Weeks after surgery
Normal group
Control group
NGF group
NGF-microsphere group
1 2 3 4
−28.22 −28.15 −28.25 −28.14
−93.32 −83.89 −68.98 −46.27
−94.95 −78.95 −58.12 −35.27
−93.22 −73.88 −50.30 −30.25
* ** ***
± ± ± ±
2.65 2.57 2.61 2.53
± ± ± ±
5.59* 4.68* 4.94* 3.15*
± ± ± ±
5.55* 4.88*,** 4.68*,** 3.22*,**
± ± ± ±
5.50* 4.82*,**,*** 4.27*,**,*** 2.43*,**,***
Compared with Normal group, p < 0.05. Compared with Control group, p < 0.05. Compared with NGF group, p < 0.05.
3.2. NGF-microsphere promotes recovery of motor function after sciatic nerve crush injury One week after injury, the SFI values in the control group, NGF group and NGF-microsphere group were in the similar range (p > 0.05, Table 2). Two to four weeks after injury, the SFI values in NGF group and NGF-microsphere group were significantly higher than that in control group (p < 0.05, Table 2). Furthermore, the SFI values in NGF-microsphere group were significantly higher than that in NGF group at 2–4 weeks (p < 0.05, Table 2), indicating that NGF-microsphere might achieve better recovery of motor function than routinely administrated-NGF after crush injury in rats. At the 4 week end point, the CMAP peak amplitude, CMAP latency of onset, and NCV values in NGF group and NGFmicrosphere group were significantly better than that in control group (p < 0.05, Table 3). In addition, all the electrophysiological values in NGF-microsphere group were significantly better than those in the NGF groups (p < 0.05, Table 3).
Four weeks after treatment, atrophy of gastrocnemius muscle was observed in control group. However, the atrophy was significantly reversed by application of NGF. The average percentage of muscle fiber area of gastrocnemius muscle in NGF group and NGFmicrosphere group were significantly better than that in control group (p < 0.05, Fig. 2). In addition, the average percentage of muscle fiber area in NGF-microsphere group was significantly better than that in NGF groups (p < 0.05, Fig. 2). 3.3. NGF-microsphere promotes recovery of sensory function after sciatic nerve crush injuries Four weeks after injury, the withdrawal threshold in NGF group and NGF-microsphere group were significantly lower than that in control group (p < 0.05, Fig. 3), indicating recovery of sensory function in NGF group and NGF-microsphere group. Furthermore, the withdrawal threshold in NGF-microsphere group were significantly lower than that in NGF group (p < 0.05, Fig. 3), indicating
Fig. 1. Representative images of transverse sectioned injured nerve under transmission electron microscope or light microscope in the Normal group (A and E), Control group (B and F), NGF group (C and G) and NGF-microsphere group (D and H) at 4 weeks after treatment. The mean diameter of the axon and the fiber was measured (i.e., the red line in A and C), and the G-ratio was calculated. Bigger diameter of the fiber might purport better nerve regeneration, and the lower G-ratio might purport thicker medulla sheath and better myelination. In A and D, the nerve fibers were in good order, the mean fiber diameter was bigger, the G-ratio was lower. The total number of myelinated axons per nerve transverse section was calculated. In E and H, the nerve fibers were in good order, and the total number of myelinated axons was larger, which might purport better nerve regeneration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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H. Yu et al. / Neuroscience Letters 566 (2014) 177–181
Table 3 The electrophysiological values at predetermined time postoperatively (n = 12).
Nerve conduction velocity (m/s) Peak amplitude of CMAP (mV) Latency of CMAP onset (ms) * ** ***
Normal group
Control group
NGF group
NGF-microsphere group
35.22 ± 3.21 41.22 ± 4.06 0.79 ± 0.072
17.23 ± 1.35* 16.12 ± 1.42* 2.15 ± 0.146*
25.87 ± 2.73*,** 24.3 ± 2.65*,** 1.72 ± 0.182*,**
33.92 ± 3.04*,**,*** 39.42 ± 3.76*,**,*** 0.88 ± 0.078*,**,***
Compared with Normal group, p < 0.05. Compared with Control group, p < 0.05. Compared with NGF group, p < 0.05.
that NGF-microsphere might achieve better recovery of sensory function than routinely administrated-NGF after crush injury in rats. Function of thermal nociceptive sensation was assessed by the hot plate test. Four weeks after injury, the TWRL values in NGF group and NGF-microsphere group were significantly lower than that in control group (p < 0.05, Fig. 3), indicating recovery of sensory function in NGF group and NGF-microsphere group. Furthermore, the TWRL values in NGF-microsphere group were significantly
lower than that in NGF group (p < 0.05, Fig. 3), indicating that NGFmicrosphere might achieve better recovery of sensory function than routinely administrated-NGF after crush injury in rats. 4. Discussion The present study investigated the possibility that local injection of controlled released NGF-microspheres might be capable of enhancing nerve regeneration and promoting functional
Fig. 2. Representative images of gastrocnemius muscle by H–E staining in the Normal group (A), Control group (B), NGF group (C), and NGF-microsphere group (D) at 4 weeks after treatment. The sum of muscle fiber cross-sectional areas in a field divided by the total area of the field multiplied by 100 (E) were shown. *Compared with Normal group, p < 0.05. # Compared with Control group, p < 0.05. ˆ Compared with NGF group, p < 0.05.
Fig. 3. Statistical analysis of mechanical withdraw threshold (A) and thermal withdraw threshold (B). *Compared with Normal group, p < 0.05; # Compared with Control group, p < 0.05; ˆ Compared with NGF group, p < 0.05.
H. Yu et al. / Neuroscience Letters 566 (2014) 177–181
recovery after sciatic nerve crush injury in rats. We found that NGF-microsphere was capable of promoting nerve regeneration and accelerating recovery of motor and sensory function after crush nerve injury. Further studies showed that local injection of controlled released NGF-microspheres achieved better nerve regeneration than intraperitoneally daily-administration of NGF. All the findings raise the possibility that controlled released NGFmicrospheres might enhance the curative effect of NGF, and might be a better neuroprotective agent for nerve crush injury. The therapy of nerve injury varied with the type of injury. For nerve section, microsurgery was generally performed for architectural reconstruction of the nerve, while drug therapy was used to promote the nerve regeneration. However, for nerve crush injury, as the continuity of the nerve was still intact, microsurgery provided little help. Drug therapy was the major therapeutic tool to promote the nerve regeneration after nerve crush injury. Although the medical science has been improved in the past decades, the outcome of nerve crush injury is unsatisfactory. As drug therapy was the major therapeutic tool to promote the nerve regeneration after nerve crush injury, increasing the curative effect of existing drugs, such as neurotrophic factors, may have more senses. NGF, as a commonly used neuroprotective agent, plays a critical role in nerve repair after injury. Previous studies proved that NGF was important for differentiation and survival of sensory and sympathetic neurons [5,17], and was cable of inducing nerve regeneration [10]. However, due to the rapid diffusion and deactivation in extracellular fluids, it is difficult for NGF to be retained at the injury sites, which limits the curative effect. In order to maintain the local concentration of NGF, a kind of controlled released NGF-microspheres was developed in our previous study [12], which was proved to be capable of enhancing peripheral nerve regeneration across short nerve gaps repaired with acellular nerve grafts. In the present study, morphometric analysis showed better nerve recovery in NGF group, and the best recovery in NGF-microsphere group. All those findings, together with the findings in the present study, highlight the beneficial effect of controlled released NGF-microspheres on nerve regeneration after nerve crush injury. Electrophysiology analysis was used to investigate the recovery of motor function after local injection of NGF-microspheres. As the CAMP amplitude is closely related to the number of nerve fibers innervating the muscle, it is possible to further evaluate the recovery of motor function [16]. In this study, better CAMP latency, NCV and CAMP peak amplitude were observed in both NGF group and NGF-microsphere group than those in the control group, indicating better recovery of motor function after NGF or NGF-microsphere treatment. Interestingly, the electrophysiological results in NGFmicrosphere group were better than those in NGF group, indicating that this controlled released system of NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration. In addition, the atrophy of the denervated muscles was partially reversed in NGF group and mostly reversed in NGF-microsphere group. The behavioral appearances were partially improved in NGF group and significantly improved in NGF-microsphere group. All these findings indicated that NGF-microspheres could enhance the beneficial effect of NGF on recovery of motor function. Further studies showed that NGF-microspheres and NGF could also improve the recovery of thermal and mechanical sensory function. Furthermore, NGFmicrospheres might result in a better recovery than NGF, indicating that NGF-microspheres could enhance the beneficial effect of NGF on recovery of sensory function. In conclusion, this controlled released NGF-microspheres could enhance the beneficial effect of NGF on nerve regeneration and recovery of motor and sensory function after nerve crush injury in
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rats, indicating that NGF-microspheres have the potential to be a neuroprotective agent for nerve injury repair applications for nerve crush injury. Further study should be done to confirm those effects in larger animals or even humans before clinical application. Competing interests The authors have declared that no competing interest exists. Acknowledgements This research was supported by the Science and Key Technology Research and Development Program of Liaoning Province (No. 2011225041 and No. 2012225019), and “Twelfth Five-Year Plan” Scientific Research Funds Project of Chinese PLA (CWS11J209). References [1] J. Noble, C.A. Munro, V.S. Prasad, R. Midha, Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries, J. Trauma 45 (July (1)) (1998) 116–122, PMID: 9680023. [2] M. Schumacher, R. Guennoun, D.G. Stein, A.F. De Nicola, Progesterone: therapeutic opportunities for neuroprotection and myelin repair, Pharmacol. Ther. 116 (October (1)) (2007) 77–106, PMID: 17659348. [3] W. Marcol, K. Kotulska, M. Larysz-Brysz, I. Matuszek, E. Olakowska, J. LewinKowalik, Extracts obtained from predegenerated nerves improve functional recovery after sciatic nerve transection, Microsurgery 25 (6) (2005) 486–494, PMID: 16134094. [4] D.J. Bryan, A.H. Holway, K.K. Wang, A.E. Silva, D.J. Trantolo, D. Wise, I.C. Summerhayes, Influence of glial growth factor and Schwann cells in a bioresorbable guidance channel on peripheral nerve regeneration, Tissue Eng. 6 (April (2)) (2000) 129–138, PMID: 10941208. [5] A.I. Gravvanis, D.A. Tsoutsos, G.A. Tagaris, A.E. Papalois, C.G. Patralexis, T.G. Iconomou, P.N. Panayotou, J.D. Ioannovich, Beneficial effect of nerve growth factor-7S on peripheral nerve regeneration through inside-out vein grafts: an experimental study, Microsurgery 24 (5) (2004) 408–415, PMID: 15378588. [6] E.G. Fine, I. Decosterd, M. Papaloïzos, A.D. Zurn, P. Aebischer, GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap, Eur. J. Neurosci. 15 (February (4)) (2002) 589–601, PMID: 11886440. [7] B. Hontanilla, C. Aubá, O. Gorría, Nerve regeneration through nerve autografts after local administration of brain-derived neurotrophic factor with osmotic pumps, Neurosurgery 61 (December (6)) (2007) 1268–1274, PMID: 18162907 (discussion 1274–1275). [8] X. Santos, J. Rodrigo, B. Hontanilla, G. Bilbao, Local administration of neurotrophic growth factor in subcutaneous silicon chambers enhances the regeneration of the sensory component of the rat sciatic nerve, Microsurgery 19 (6) (1999) 275–280, PMID: 10469442. [9] R. Ikeguchi, R. Kakinoki, T. Matsumoto, T. Yamakawa, K. Nakayama, Y. Morimoto, H. Tsuji, J. Ishikawa, T. Nakamura, Basic fibroblast growth factor promotes nerve regeneration in a C− -ion-implanted silicon chamber, Brain Res. 1090 (May (1)) (2006) 51–57, PMID: 16677621. [10] S.D. Skaper, Nerve growth factor: a neurokine orchestrating neuroimmuneendocrine functions, Mol. Neurobiol. 24 (August–December (1–3)) (2001) 183–199, PMID: 11831552. [11] C.C. Tsai, M.C. Lu, Y.S. Chen, C.H. Wu, C.C. Lin, Locally administered nerve growth factor suppresses ginsenoside Rb1-enhanced peripheral nerve regeneration, Am. J. Chin. Med. 31 (5) (2003) 665–673, PMID: 14696670. [12] H. Yu, J. Peng, Q. Guo, L. Zhang, Z. Li, B. Zhao, X. Sui, Y. Wang, W. Xu, S. Lu, Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor, Microsurgery 29 (4) (2009) 330–336, PMID: 19296502. [13] J.R. Bain, S.E. Mackinnon, D.A. Hunter, Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat, Plast. Reconstr. Surg. 83 (January (1)) (1989) 129–138, PMID: 2909054. [14] C.F. Vogelaar, D.H. Vrinten, M.F. Hoekman, J.H. Brakkee, J.P. Burbach, F.P. Hamers, Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome, Brain Res. 1027 (November (1–2)) (2004) 67–72, PMID: 15494158. [15] J. Ma, W. Li, R. Tian, W. Lei, Ginsenoside Rg1 promotes peripheral nerve regeneration in rat model of nerve crush injury, Neurosci. Lett. 478 (2) (2010) 66–71, PubMed: 20438804. [16] X. Hu, J. Huang, Z. Ye, L. Xia, M. Li, B. Lv, X. Shen, Z. Luo, A novel scaffold with longitudinally oriented microchannels promotes peripheral nerve regeneration, Tissue Eng. A 15 (November (11)) (2009) 3297–3308, PMID: 19382873. [17] L. Aloe, Rita Levi-Montalcini: the discovery of nerve growth factor and modern neurobiology, Trends Cell Biol. 14 (July (7)) (2004) 395–399, PMID: 15246433.
