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Rat Nerve Regeneration with the Use of a Polymeric Membrane Loaded with NGF

¹ Faculté de Chirurgie Dentaire, Université de Lille 2, Place de Verdun, 59000 Lille, France;
² Unité de Neurosciences et Physiologie Adaptatives, UPRES EA 4052, Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France; and
³ INSERM Unité 595, Faculté de Chirurgie Dentaire, Université Louis Pasteur, 11 rue Humann, 67085 Strasbourg Cedex, France

Correspondence: ^* corresponding author, mathilde.savignat{at}univ-lille2.fr

	ABSTRACT

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Exogenous neurotrophic factors, delivered by various systems, are used to improve nerve regeneration. This study tested the effectiveness of a polymeric membrane loaded with Nerve Growth Factor (NGF) on mental nerve regeneration after a crush injury in rats. We tested NGF application, known to play a role in afferent fiber repair in dental neurobiology, to see if it could improve the regeneration. Afferent neurogram recordings and histological analyses of the trigeminal ganglion neurons were performed. One month after the crush injury, early regeneration was observed independently of exogenous NGF. However, as compared with the activity level recorded before the injury, the afferent activity was reduced by 28.5% without NGF, and the mean number of labeled neurons decreased. With NGF, activity was increased by 30.8%, with no significant histological difference compared with animals without lesions. NGF application through a polymeric membrane can influence degenerative and/or regenerative processes after a crush injury.

Key Words: lower lip • neurotrophin • polymeric films • afferent neurograms • trigeminal ganglion

	INTRODUCTION

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The inferior alveolar nerve is frequently damaged during surgical procedures involving the mandible (Strauss et al., 2006), particularly during third molar extractions (Sandstedt and Sörensen, 1995; Loescher et al., 2003). Injuries can give rise to persistent paresthesia, dysesthesia, and pain (Robinson, 1988; Hildebrand et al., 1995; Sandstedt and Sörensen, 1995). Patients may benefit from surgical intervention (Loescher et al., 2003), such as suturing and/or nerve grafts (Holland, 1996; Robinson et al., 2004; Strauss et al., 2006). These are delicate procedures, however, and are ineffective in cases of crush injuries, one of the most common lesions. Studies of biological or synthetic conduits that release neurotrophic factors have also been reported in the literature (Frostick et al., 1998; Santos et al., 1998; Rosner et al., 2003).

Recently, polymeric films have emerged as a new method of delivering biomolecules (Berg et al., 2006). For this study, we developed new membranes with polymeric films containing a neurotrophic factor to regenerate rat nerves. It has been demonstrated in vitro that the viability of motoneurons was increased by these polymeric multilayers (Vodouhe et al., 2005). However, to the best of our knowledge, the impact of these multilayered films had never been evaluated in vivo.

Nerve growth factor (NGF) is required by the nociceptive neurons (McMahon et al., 1994) of the dental pulp (Hildebrand et al., 1995; Fried et al., 2000). NGF promotes the development, survival, and regeneration of the dental sensory neurons (Buchman and Davies, 1993; Naftel et al., 1994; Qian and Naftel, 1996; Huang et al., 1999). NGF also has a chemoattractant effect in vitro on Schwann cells (Maniwa et al., 2003), which play a crucial role in nerve regeneration (Frostick et al., 1998).

Based on theses studies, it was postulated that exogenous NGF, a neurotrophin known to influence afferent fiber regeneration, when embedded in our polymeric membrane, could improve mental nerve regeneration after a crush injury in rats.

	MATERIALS & METHODS

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Animal Groups
Male Wistar rats (C. Rivers-Iffa Credo–l’Arbresle, France) were used, each weighing 280–300 g. Testing followed a protocol to ensure humane practices, as recommended by the Agricultural and Forest Ministry and the National Education Ministry (B59-00913).

Electrophysiology
Injured rats without the membrane (CONT, n = 10), with the membrane but without NGF (SHAM, n = 10), and with the membrane loaded with NGF (NGF, n = 10) were used. A separate test group of rats was operated on without any injury or membrane treatment (TEM, n = 4).

Histological Analysis
We used non-injured control rats (TEM, n = 7), injured rats without the membrane after 1 (CONT 1 mo, n = 6) and 3 mos (CONT 3 mos, n = 5), and injured rats with the membrane loaded with NGF after 1 (NGF 1 mo, n = 7) and 3 mos (NGF 3 mos, n = 5).

Build-up of PLGA Membranes and Coatings with Polymeric Multilayers
Poly(D,L-lactic-co-glycolic acid) copolymer, ratio 50:50 (PLGA, Mw = 4 x 10⁴ – 7.5 x 10⁴ Da), purchased from Sigma (St. Quentin Fallavier, France), was dissolved in chloroform at room temperature at 10 mg/mL. PLGA membranes were then built up by the solvent-casting method. Poly(L-lysine) (PLL, Mw = 5.7 x 10⁴ Da) and poly(D-glutamic acid) (PGA, Mw = 5.0 x 10⁴ Da - 1.0 x 10⁵ Da) were purchased from Sigma and prepared at 1 mg/mL by dissolution in 0.15 M NaCl solution (pH = 5.9). To build the multilayers, we first dipped the PLGA membranes in a polycation solution (PLL) for 10 min. Then, we performed a rinsing step by dipping the substrates for 10 min in 0.15 M NaCl solution. The polyanion (PGA) was deposited in the same manner. The build-up process was continued by the alternating deposition of PLL and PGA. After the deposition of n bilayers, the film is denoted (PLL/PGA)_n. In the case of membranes loaded with Human β-Nerve growth factor (NGF), NGF (40 µg/mL in NaCl 0.15 M) was adsorbed in the same way, except that PGA was replaced by NGF at a certain stage of the build-up process, and with a longer time of adsorption (3 hrs). The amount of NGF embedded was determined by a previously published method (Tezcaner et al., 2006). Finally, architectures obtained corresponded to (PLL/PGA)₂/PLL/NGF/ (PLL/PGA)/PLL/NGF/(PLL/PGA)PLL/NGF multilayers.

Electrophysiological Study
With the rats under sodium pentobarbital anesthesia (30 mg/kg), a submandibular skin incision was made. Another incision was performed to allow access to the mental foramen.

Afferent neurograms were recorded by means of a 100-µm unipolar needle electrode, which impaled the mental nerve posterior to the lesion, near the mental foramen. For each rat, afferent activity was recorded before the nerve was crushed. The injury was created with the use of ultrafine forceps. The lesion was immediately verified based on the absence of afferent action potential after stimulation of the ipsilateral labial commissura with a vessel clip. This stimulation was chosen because it was the most responsive and accessible region of the ipsilateral lower lip. The vessel clip exerted a pressure of 54 mN/mm². Twenty four-second-long stimulations were applied, each followed by 40 sec of rest. All afferent neurograms were recorded during a 20-minute period.

Polymeric membrane, 3 mm wide and 6 mm long, either loaded or not with 234 ng NGF, was then placed around the nerve at the site of the lesion and fixed with cyanoacrylate glue (see APPENDIX). After surgery, the incision was sutured, and betadine (betadine, polyvidone iodée, Viatris, Merignac, France) was applied. The animals were maintained under analgesic (Metacam: 100 µL/kg, Boehringer Ingelheim, Germany) and antibiotic (Trisulmix liquid: 100 µL/kg, Coophavet, France) treatment for 3 and 7 days, respectively.

Neurograms were recorded 1, 2, and 3 mos after the crush injury.

Recording and Analysis
Nerve activity was amplified 20,000 (pre-amplifier model P 511, Grass Instruments, Quincy, MA, USA; band pass, 30 Hz to 3 kHz), recorded, and analyzed through interactive software (SPIKE 2, Cambridge Electronic Design, Cambridge, UK). Afferent neurograms were rectified throughout the recorded stimulation periods. The integrated neurogram values were then normalized with regard to the noise. For each rat, the 20 afferent bursts, recorded after each stimulation, were pooled and averaged. When the experiment was complete, the rats were given a lethal dose of sodium pentobarbital.

Histological Analysis of Neuron Soma in the Trigeminal Ganglion
Rats were anesthetized with sodium pentobarbital (30 mg/kg). Seven days before the end of the experiment, a retrograde neuron labeling, with 3 injections of 5 µL of a fluorescent tracer [1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate: FAST DiI^TM, DiI^9,12 -C₁₈(3),CBS; D 7756, Invitrogen, Leiden, Netherlands] was carefully administered to each rat in the ipsilateral lower lip by means of a Hamilton syringe.

Seven days later, the animals were transcardially perfused with saline and paraformaldehyde (PFA) solutions (De-Doncker et al., 2006). After craniotomy, the ipsilateral trigeminal ganglia were removed and post-fixed overnight with the same solution of PFA. The ganglia were immersed in a 20% sucrose solution for 4 days, then embedded in Tissue Tek (Sakura, Tokyo, Japan) and frozen in liquid nitrogen. Serial 35-µm longitudinal sections were made at –20°C in a cryostat microtome (LEICA CM 1800, Heidelberg, Germany). Sections were then observed under a fluorescence microscope equipped with a rhodamine filter (Axioplan 2 imaging, ZEISS, Oberkochen, Germany). Only neurons with a visible nucleus, bright fluorescence, and well-delimited soma were analyzed. The numbers and soma sizes (µm²) of neurons were measured with ANALYSIS software.

Statistical Analysis
Soma size distribution histograms for each rat in each histological group were created with SYSTAT 11.0 software, and their medians were determined. Histogram medians were compared by the Kruskal-Wallis test (P < 0.05). The two-sample Kolmogorov-Smirnov’s test was used to compare the global distribution histogram of neurons between the CONT and NGF groups.

Electrophysiological results were expressed as means ± SD. We used a one-way ANOVA and Bonferroni t test to observe significant differences (P < 0.05).

	RESULTS

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For all animals, no inflammatory responses, edema, or infections at 1 and 3 mos were observed, and the membrane was degraded after 3 mos.

Electrophysiological Data
Before and Immediately After the Crush Injury
After mechanical stimulations, action potential bursts appeared in the CONT and NGF groups, but were not maintained during the entire stimulation period (Fig. 1A). Activity disappeared immediately after the lesion was created (Fig. 1B).

View larger version (36K): [in this window] [in a new window]   Figure 1. Qualitative representations of the ipsilateral mental nerve afferent neurograms before (A), immediately after (B), 1 mo after (C), and 3 mos after (D) a crush lesion in the CONT and NGF groups. The horizontal hatched line indicates the four-second-long stimulation of the ipsilateral labial commissura. Note the increased activity with NGF treatment at 1 mo after lesion creation.

View larger version (41K): [in this window] [in a new window]   Figure 2. Histogram of quantitative adaptive changes in the mental nerve afferent neurograms before and 1, 2, and 3 mos after a crush lesion in the CONT (n = 10, black bars), SHAM (n = 10, white bars), and NGF (n = 10, hatched bars) groups. Results are expressed as mean ± SD (V). Gain = 20,000. Note the significant increase in the afferent activity, 1 mo after the crush, with NGF treatment. *Indicates a significant difference compared with the CONT group, +difference compared with the SHAM group, and #difference compared with the value before the lesion in each group (P < 0.05).

Histological Analysis
Because no electrophysiological difference was observed between the CONT and SHAM groups, histological analysis was not performed on the SHAM group. An example of retrograde ganglion labelings is illustrated in Fig. 3. In total, 14,085 sensory neurons were analyzed.

View larger version (26K): [in this window] [in a new window]   Figure 3. Isolated rat trigeminal ganglion (A) and sensory neuron soma labelings in the mandibular division (B) after FAST DiI injections in the ipsilateral lower lip.

Non-injured Mental Nerve
For the TEM group, the mean number of labeled sensory neurons in the ipsilateral trigeminal ganglion was 498.6 ± 74.2. The mean median of soma sizes was 895.2 ± 80.2 µm², close to that of the global distribution histogram (Fig. 4).

View larger version (26K): [in this window] [in a new window]   Figure 4. Global distribution histograms of soma sizes (µm2) of the total number of labeled sensory neurons in each animal group: TEM, 1 mo (A; n = 7 rats and 3490 neurons); CONT, 1 mo (B; n = 6 rats and 1950 neurons); and NGF, 1 mo (C; n = 7 rats and 3630 neurons).

Three Months after Lesion Creation
No significant difference was observed in the CONT and NGF (3 mos) groups in the mean numbers of neurons (493 ± 81.7 and 510 ± 87.7, respectively), mean medians of soma sizes (888 ± 47.5 µm² and 908.4 ± 18.5 µm², respectively), and the medians of the global distribution histogram (897.2 µm² and 904 µm², respectively), in comparison with the TEM group.

	DISCUSSION

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The application of a single low dose of exogenous NGF through a novel polymeric membrane did have an effect on degenerative and/or regenerative processes after a crush injury.

The deafferentation of the lower lip, the appearance of spontaneous activity, and the regeneration kinetics observed in this study were consistent with those of previous work on regeneration after inferior alveolar nerve or mental nerve injuries in rats (Berger et al., 1983; Chudler et al., 1997; Verze et al., 2003). In our results, the partial recovery of activity in the absence of exogenous NGF was due, in part, to the regeneration of a fraction of sensory neurons. Indeed, there was good correspondence between the afferent activity decrease and the 34.8% decrease in the mean number of regenerated neurons. The fact that 65.2% of neurons regenerated in the CONT (1 mo) group, in spite of the lack of exogenous NGF, could suggest that the majority of lower lip axons were myelinated non-NGF-dependent neurons of large to medium diameters, which recovered more readily (Holland, 1996; Imai et al., 2003). The increase in the distribution histogram median of the CONT group in comparison with that of the non-injured TEM group is consistent with this interpretation. Moreover, Naftel et al.(1999) showed that 68% of mental nerve axons in rats were myelinated, the remaining 32% being non-myelinated, nociceptive axons, presumably NGF-dependent (McMahon et al., 1994).

Numerous studies have attempted to find a system capable of delivering neurotrophin at the injury site (see APPENDIX). However, problems remain: the necessity to refill or remove the neurotrophic solution to maintain bioactivity; the presence of infections due to partial biocompatibility and high numbers of surgical procedures; and the need to maintain support in the correct position for the necessary amount of time (Santos et al., 1998; Rosner et al., 2003). The production and requirements of neurotrophic factors by the regenerating nerve are not constant during the recovery time after lesion creation (Windebank and Poduslo, 1986; Boyd and Gordon, 2003). The critical period is during the 1st wk and extends through the 4th or 8th wk (depending on lesion type), when the endogenous neurotrophic factor concentration is low, whereas the neurons strongly express NGF receptors. It is during this critical period that NGF administration is most effective (Oudega and Hagg, 1996). Consequently, the challenge is to deliver the neurotrophic substance at correct doses and rates at the injury site, while maintaining bioactivity by protecting the factor against biodegradation mechanisms.

In our study, afferent activity measured 1 mo after lesion creation was higher in the NGF group than in the CONT group. This increase was due, in part, to the regeneration of the 34.8% of the remaining axons, presumably small to medium-sized NGF-dependent neurons. However, this alone does not explain the 30.8% increase in activity beyond the level recorded before the crush injury that was seen in the NGF group. It is possible that the NGF could induce a hypersensitivity of the sensory neurons. Indeed, in several rat studies, NGF induced mechanical hyperalgesia (Lewin et al., 1993; Stucky et al., 1999). In the non-injured animals, the nociceptive fibers were probably not affected by our mechanical stimulation, since the majority of nociceptors of the rat oral mucosa have mechanical thresholds above 54 mN/mm² (Toda et al., 1997). In the NGF group, nociceptive fibers, which are NGF-dependent (Lewin et al., 1993; Mendell et al., 1999; Stucky et al., 1999), were probably recruited by our stimulation. Although it is difficult to determine the effect of this mechanical sensitization in activity recovery, it is demonstrated that prevailing nerve regeneration occurred, as assessed by the retrograde labeling of sensory neurons.

Our polymeric membrane is an entirely biocompatible and biodegradable surface. Although the release rate and the exact dose of NGF delivered by the membrane are unknown, one can conclude, from the results of this study, that a single, low dose of 234 ng of exogenous NGF enhances regeneration throughout the 1st mo after lesion creation, when the concentration of endogenous NGF is low. The results of this study also raise the possibility that NGF could affect the nerve degeneration process. The use of this polymeric membrane could have important therapeutic implications for nerve regeneration after a crush injury.

	ACKNOWLEDGMENTS

 
Experiments were performed in the Laboratoire de Plasticité Neuromusculaire, UPRES EA 4052, USTL, Villeneuve d’Ascq, France, directed by Professor Falempin. They were supported by the Conseil Régional du Nord-Pas-de-Calais.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received for publication October 4, 2006. Revision received June 11, 2007. Accepted for publication June 15, 2007.

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Journal of Dental Research, Vol. 86, No. 11, 1051-1056 (2007)
DOI: 10.1177/154405910708601106


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This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Savignat, M. Articles by Libersa, P. Search for Related Content PubMed PubMed Citation Articles by Savignat, M. Articles by Libersa, P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB LACTIC ACID Social Bookmarking                   What's this?