Peripheral Nerve Regeneration

Photomed Laser Surg. 2016 Dec;34(12):638-645. doi: 10.1089/pho.2016.4095.

Photobiomodulation Triple Treatment in Peripheral Nerve Injury: Nerve and Muscle Response.

Mandelbaum-Livnat MM1, Almog M1, Nissan M1, Loeb E2, Shapira Y1, Rochkind S1.

Author information

  • 11 Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University , Tel Aviv, Israel .
  • 22 Pharmaseed , Ness Ziona, Israel .

Abstract

BACKGROUND:

Muscle preservation or decrease in muscle degeneration and progressive atrophy are major challenges in patients with severe peripheral nerve injury (PNI). Considerable interest exists in the potential therapeutic value of laser phototherapy (photobiomodulation) for restoring denervated muscle atrophy and for enhancing regeneration of severely injured peripheral nerves. As previously published, the laser phototherapy has a protective and immediate effect in PNI. Laser phototherapy in the early stages of muscle atrophy may preserve the denervated muscle by maintaining creatinine kinase (CK) activity and the amount of acetylcholine receptor (AChR).

OBJECTIVE AND METHODS:

In the present study, the effectiveness of triple treatment laser phototherapy, namely, applied simultaneously at three areas: injured area of the peripheral nerve, corresponding segments of the spinal cord, and corresponding denervated muscle (triple treatment), was evaluated for the treatment of incomplete PNI in rats with the ultimate goal of achieving improved limb function.

RESULTS:

Forty-five days after the sciatic nerve insult, all rats regained normal walking (functional sciatic index values returned to baseline); however, the long laser irradiation (7?min) group presented the fastest recovery as opposed to short laser irradiation (3?min). A histological evaluation of the nerves revealed that long laser irradiation led to a higher amount of neuronal fibers that were larger than 4??m (543?±?76.8, p?<?0.01) than short irradiation (283?±?35.36). A histological evaluation of muscular atrophy showed that long laser irradiation evolved with significantly less muscle atrophy (8.06%?±?1.23%, p?<?0.05) than short irradiation (24.44%?±?7.26%).

CONCLUSIONS:

The present study and our previous investigations showed that the laser phototherapy increases biochemical activity and improves morphological recovery in muscle and, thus, could have direct therapeutic applications on muscle, especially during progressive atrophy resulting from PNI.

Lasers Med Sci. 2016 Jul;31(5):965-72. doi: 10.1007/s10103-016-1939-2. Epub 2016 Apr 25.

The new heterologous fibrin sealant in combination with lowlevel laser therapy (LLLT) in the repair of the buccal branch of the facial nerve.

Buchaim DV1, Rodrigues Ade C2, Buchaim RL2, Barraviera B3, Junior RS3, Junior GM4, Bueno CR4, Roque DD5, Dias DV6, Dare LR6, Andreo JC2.

Author information

  • 1Human Morphophysiology (Anatomy), Faculty of Medicine, University of Marilia (UNIMAR), Marilia, SP, Brazil. danibuchaim@usp.br.
  • 2Department of Biological Sciences (Anatomy), Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil.
  • 3Center for the Study of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP-Univ Estadual Paulista), Botucatu, São Paulo State, Brazil.
  • 4University of the Sacred Heart, Bauru, SP, Brazil.
  • 5Human Morphophysiology (Anatomy), Faculty of Medicine, University of Marilia (UNIMAR), Marilia, SP, Brazil.
  • 6Federal University of Pampa-UNIPAMPA, Uruguaiana, RS, Brazil.

Abstract

This study aimed to evaluate the effects of lowlevel laser therapy (LLLT) in the repair of the buccal branch of the facial nerve with two surgical techniques: end-to-end epineural suture and coaptation with heterologous fibrin sealant. Forty-two male Wistar rats were randomly divided into five groups: control group (CG) in which the buccal branch of the facial nerve was collected without injury; (2) experimental group with suture (EGS) and experimental group with fibrin (EGF): The buccal branch of the facial nerve was transected on both sides of the face. End-to-end suture was performed on the right side and fibrin sealant on the left side; (3) Experimental group with suture and laser (EGSL) and experimental group with fibrin and laser (EGFL). All animals underwent the same surgical procedures in the EGS and EGF groups, in combination with the application of LLLT (wavelength of 830 nm, 30 mW optical power output of potency, and energy density of 6 J/cm(2)). The animals of the five groups were euthanized at 5 weeks post-surgery and 10 weeks post-surgery. Axonal sprouting was observed in the distal stump of the facial nerve in all experimental groups. The observed morphology was similar to the fibers of the control group, with a predominance of myelinated fibers. In the final period of the experiment, the EGSL presented the closest results to the CG, in all variables measured, except in the axon area. Both surgical techniques analyzed were effective in the treatment of peripheral nerve injuries, where the use of fibrin sealant allowed the manipulation of the nerve stumps without trauma. LLLT exhibited satisfactory results on facial nerve regeneration, being therefore a useful technique to stimulate axonal regeneration process.

Support Care Cancer. 2016 Jan;24(1):233-42. doi: 10.1007/s00520-015-2773-y. Epub 2015 May 26.

Lowlevel laser therapy alleviates mechanical and cold allodynia induced by oxaliplatin administration in rats.

Hsieh YL1, Fan YC1, Yang CC2.

Author information

  • 1Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan.
  • 2Department of Physical Medicine and Rehabilitation, Cheng Ching General Hospital, Taichung, Taiwan. s901100@gmail.com.

Abstract

PURPOSE:

Cold and mechanical allodynia caused by oxaliplatin-induced acute peripheral neuropathy frequently occur after drug infusion. Lowlevel laser therapy (LLLT) has been used to improve pain symptoms associated with various conditions and may have potential as a therapy for oxaliplatin-induced allodynia. The purpose of the present study was to investigate the antiallodynic effect of LLLT in an oxaliplatin-treated animal model by assessing sensory behavioral responses, levels of nerve growth factor (NGF), and transient receptor potential M8 (TRPM8) in dorsal root ganglia (DRG) neurons, as well as substance P (SP) in the spinal dorsal horn.

METHODS:

Adult male Sprague-Dawley rats each received a total of four doses of oxaliplatin (4 mg/kg, i.p.), injected at 3-day intervals. Following oxaliplatin administration, LLLT (7.5 J/cm(2)) was applied for 12 consecutive days to the skin surface directly above sites where the sciatic nerve is distributed. Behavioral assessments were then performed, followed by immunoassays for NGF, TRPM8, and SP proteins.

RESULTS:

LLLT relieved both cold and mechanical allodynia induced by oxaliplatin in rats. Oxaliplatin-related increases in protein levels of NGF and TRPM8 in DRG and SP in the dorsal horn were also reduced after LLLT.

CONCLUSION:

The findings of this study support LLLT as a potential treatment for oxaliplatin-induced neuropathy. Moreover, our findings suggest that SP, TRPM8, and NGF proteins in the superficial dorsal horn and DRG may be involved in an antiallodynic effect for LLLT

PLoS One. 2014; 9(8): e103348.
Published online 2014 Aug 13. doi:  10.1371/journal.pone.0103348
PMCID: PMC4131879

Low-Level Laser Irradiation Improves Functional Recovery and Nerve Regeneration in Sciatic Nerve Crush Rat Injury Model

Chau-Zen Wang,1,2 Yi-Jen Chen,3,4 Yan-Hsiung Wang,2,5 Ming-Long Yeh,6 Mao-Hsiung Huang,3,7 Mei-Ling Ho,1,2 Jen-I Liang,6 and Chia-Hsin Chen2,3,4,7,*
Michael Hamblin, Editor
1Department of Physiology, Kaohsiung Medical University, Kaohsiung, Taiwan
2Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan
3Department of Physical Medicine and Rehabilitation, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
4Department of Physical Medicine and Rehabilitation, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
5School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
6Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan
7Department of Physical Medicine and Rehabilitation, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
MGH, MMS, United States of America
Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: CZW YHW CHC. Performed the experiments: CZW YJC MLY JIL CHC. Analyzed the data: CZW YHW MHH MLH CHC. Contributed reagents/materials/analysis tools: CZW YHW YJC MLY CHC. Contributed to the writing of the manuscript: CZW YJC YHW CHC.

Author information ? Article notes ? Copyright and License information ?
Received 2014 Apr 6; Accepted 2014 Jun 27.

Abstract

The development of noninvasive approaches to facilitate the regeneration of post-traumatic nerve injury is important for clinical rehabilitation. In this study, we investigated the effective dose of noninvasive 808-nm low-level laser therapy (LLLT) on sciatic nerve crush rat injury model. Thirty-six male Sprague Dawley rats were divided into 6 experimental groups: a normal group with or without 808-nm LLLT at 8 J/cm2 and a sciatic nerve crush injury group with or without 808-nm LLLT at 3, 8 or 15 J/cm2. Rats were given consecutive transcutaneous LLLT at the crush site and sacrificed 20 days after the crush injury. Functional assessments of nerve regeneration were analyzed using the sciatic functional index (SFI) and hindlimb range of motion (ROM). Nerve regeneration was investigated by measuring the myelin sheath thickness of the sciatic nerve using transmission electron microscopy (TEM) and by analyzing the expression of growth-associated protein 43 (GAP43) in sciatic nerve using western blot and immunofluorescence staining. We found that sciatic-injured rats that were irradiated with LLLT at both 3 and 8 J/cm2 had significantly improved SFI but that a significant improvement of ROM was only found in rats with LLLT at 8 J/cm2. Furthermore, the myelin sheath thickness and GAP43 expression levels were significantly enhanced in sciatic nerve-crushed rats receiving 808-nm LLLT at 3 and 8 J/cm2. Taken together, these results suggest that 808-nm LLLT at a low energy density (3 J/cm2 and 8 J/cm2) is capable of enhancing sciatic nerve regeneration following a crush injury.

Introduction

Peripheral nerve injury results from various etiologies, such as traction, crushing, ischemic change, cutting injury and long bone fracture, that lead to axonotmesis, in which axons and the covering myelin sheaths are damaged but the connective tissue is preserved, or more severely, neurotmesis, which involves disruption of the entire nerve fiber [1], [2]. Injury to the peripheral nerve results in secondary muscle atrophy, causing various levels of disabilities. Once the peripheral nerve is damaged, degeneration occurs both distal to the injured site through Wallerian degeneration and proximal to the injured site through retrograde degeneration, influencing the corresponding neurons [3]. The sciatic nerve is a large nerve fiber that originates from the lumbosacral plexus with mixed motor and sensory components and is responsible for the motor control and sensory innervation of the lower limbs. Trauma, entrapment and ischemia can cause sciatic nerve damage and lead to limb dysfunction.

Regeneration occurs, albeit slowly, after peripheral nerve injury. Surgical repair is the mainstay for severe or complete nerve injury. Surgical approaches have been developed to repair injured nerves using advanced techniques, such as allograft, autograft and emerging materials science and engineering techniques [4], [5]. Nevertheless, non-surgical approaches have also been developed to facilitate nerve regeneration either for the primary management of axonotmesis or as an adjunctive therapy after surgical repair. The foremost treatment of nerve injury should be rehabilitation programs to maintain adequate joint range of motion and muscle tone to avoid secondary muscle atrophy. Physical therapies such as low frequency electrical stimulation [6][8] and magnetic stimulation [9] were proposed to have positive effects on nerve regeneration and functional recovery. Growth factors and neural stem cell transplantation were also claimed to have important neuroprotective effects [10], [11]. However, the high medical expense and invasiveness of these procedures prevents their use in routine clinical practice.

Low-level laser therapy (LLLT) was introduced into medical applications in the 1960s. It is a noninvasive treatment modality that has been applied in various fields, is effective in pain relief and promotes the recovery of some pathologies, including tendinopathies, osteoarthritis, rheumatoid arthritis, wound healing and nerve injuries [12][15]. Previous studies have shown positive biological effects of low-level laser stimulation on the nervous system. Several randomized controlled trials applying a low-level laser to an injured peripheral nerve show positive effects with respect to accelerating regenerative processes after the injury [16], [17]. Improved peripheral nerve function leading to significant functional recovery following LLLT was also proposed by Rochkind et al [18].

Animal models of peripheral nerve injury have been developed to evaluate the effect of LLLT in the regeneration of peripheral nerve injury [19][21]. Functional, histological, morphological and electrophysiological assessments of the effect of LLLT proved that it had beneficial effects on the regeneration of rat sciatic nerve following an injury [20][23]. Shin et al. [24] also found elevated GAP43 immunoreactivity in regenerating peripheral nerves after LLLT, suggesting more rapid neural recovery. Morphologic changes evaluated by light microscopy and electron microscopy were also used to determine the extent of demyelination and vascular changes in the peripheral nerve segment [25], [26]. However, a thorough evaluation of the effects of LLLT using molecular, histological and morphological analyses to assess functional recovery has not been performed, and the optimal parameters of LLLT to facilitate peripheral nerve regeneration are still controversial. The purpose of this study is to determine the effects of LLLT and the effective laser dose to facilitate neural regeneration in a rat sciatic nerve injury model, using both molecular and functional assessments.

Materials and Methods

Sciatic nerve crush injury in rat

Sprague Dawley (SD) rats were purchased from the National Laboratory Center (Taipei, Taiwan), and the in vivo experiments were performed with the approval of the Kaohsiung Medical University Animal Care and Use Committee. A total of 36 9-week-old SD rats were randomly divided into 6 experimental groups: (1) a normal group without 8 J/cm2 LLLT (Normal), (2) a normal group with 8 J/cm2LLLT, in which the animals received no sciatic nerve crush (Normal+8J), (3) a sciatic nerve crush group, in which the animals received a right sciatic nerve crush, without LLLT (Crush), (4) a sciatic nerve crush group with 3 J/cm2 LLLT (Crush+3J), (5) a sciatic nerve crush group with 8 J/cm2 LLLT (Crush+8J) and (6) a sciatic nerve crush group with 15 J/cm2 LLLT (Crush+15J). The food was limited to 15 g per rat every day. To create the sciatic nerve crush injury in the right hindlimb, the rats were anesthetized by intraperitoneal injection of ketamine (Ketalar, Parke-Davis, Taiwan) in combination with xylazine-hydrochloride (Rompun, Bayer HealthCare, Germany). The hair over the posterior hindlimb was shaved and cleaned with a depilator. The skin was sterilized with 10% povidone iodine and then incised 2 cm open over the posterior hindlimb to expose the sciatic nerve. A non-serrated forceps with smoothed surface was used to create a 3-mm-long crush injury at the right sciatic nerve by approximately 54 N for 30 seconds, and the left hindlimb was a sham control surgery without crush injury. The wounds were sutured after surgery with nylon 5-0 sutures. For the LLLT, the rats were treated daily with a total energy of 3, 8 or 15 J/cm2 laser irradiation to the center of the surgical site. After twenty days of LLLT, the rats were euthanized using CO2 inhalation. The sciatic nerve was quickly removed, embedded in Tissue-Tek OCT compound 4583 (Sakura Finetech, Tokyo, Japan) and frozen in liquid nitrogen. The 5-µm-thick sections of sciatic nerve were cut serially in a cryostat and collected onto glass slides (MAS-GP type A; Matsunami Glass, Osaka, Japan) for immunofluorescence analysis.

Low-level laser therapy (LLLT)

The Gallium Aluminum Arsenide (Ga-Al-As) near-infrared laser with a wavelength of 808 nm (Transverse IND. CO., LTD., Taipei, Taiwan) was used as the laser source at a laser power output of 170 mW and power density of 44.7 mW/cm2. After sciatic nerve crush injury of the rat’s right hindlimb, 808-nm LLLT was applied over the sciatic nerve crush site, and a total energy of 3, 8 or 15 J/cm2 on the surface of the skin was applied once per day for 20 consecutive days in each intervention group. The laser irradiation parameters are listed in Table 1. The sham-operated sciatic nerve of the rat left hindlimb was subjected to LLLT as indicated.

Table 1

The Laser Irradiation Parameters.

Sciatic functional index (SFI) analysis for functional assessment of sciatic nerve regeneration

The SFI was used as described by De Medinaceli et al [27] to assess the functional recovery of the damaged rat sciatic nerve by comparing the SFI before LLLT and after 20 days of LLLT. The assessment occurred on a transparent acrylic track that was 80 cm long and 6 cm wide with a small, dark shelter at the end. The rats were walked down the track and real-time images were captured via digital camera recording. Then, the footprint images were evaluated using the computer program ImageJ. The SFI was calculated according to the following equation as described by Bain et al [28]:

SFI?=??38.3 [(EPL-NPL)/NPL]+109.5 [(ETS-NTS)/NTS]+13.3 [(EITS-NITS)/NITS]?8.8. The print length (PL, the distance from the heel to the third toe), toe spread (TS, distance from the first to the fifth toe) and intermediary toe spread (ITS, distance from the second to the fourth toe) of the experimental (E) and normal (N) sides were measured from the recorded footprints in a gait track.

Range of motion (ROM) analysis for functional assessment of sciatic nerve regeneration

Functional assessments of nerve regeneration by ROM were measured to evaluate the degree of functional recovery before and after LLLT. After 20 days, the ROM of the experimental (right hindlimb) and control (left hindlimb) hind feet was measured during the mid-stance phase of the gait cycle.

Transmission electron microscopy (TEM) analysis for evaluation of the myelin sheath thickness in sciatic nerve

The TEM imaging grids were prepared by dropping centrifuged solutions onto a carbon-coated copper grid followed by drying. TEM images of the sciatic nerve were captured and conducted using a JEM-2000EXII transmission electron microscope (Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) operating at 200 kV. An energy dispersive spectroscopy system (Link II Energy Dispersive X-ray Analysis System, England) attached to the microscope was used to determine the chemical characterization of the sample. The myelin sheath thickness in the TEM images was measured using an image analysis system (Image-Pro Plus; Media Cybernetics Inc., Silver Spring, MD, USA).

Western blotting analysis for detecting the GAP43 expression in sciatic nerve

A sciatic nerve segment with its proximal 1 cm and distal 1 cm lengths was obtained. The sciatic nerve segment was washed twice with ice-cold PBS containing 1 mM sodium vanadate and lysed in a modified radio immunoprecipitation assay buffer (RIPA: 150 mM NaCl, 1 mM EGTA, 50 mM Tris [pH 7.4], 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) with protease inhibitors (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics Ltd., Taipei, Taiwan) and 1 mM sodium vanadate. The lysates were cleared using centrifugation at 14,000 rpm for 15 min at 4°C and analyzed using western blotting with anti-GAP43 (Abcam, Cambridge, MA) and anti-?-actin (Sigma-Aldrich, St. Louis, MO) antibodies. The membranes were developed using the Immobilon Western HRP Substrate (Millipore, Billerica, MA). The blots were digitally evaluated and measured using the UVP AutoChemi™ Image and Analysis System (UVP, Upland, CA).

Immunofluorescence for detecting GAP43 and neurofilament in sciatic nerve

The 5-µm-thick sections of sciatic nerve on glass slides were washed three times with PBS and then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After washing three times with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, rinsed with PBS and then immunostained with anti-GAP43 (Abcam) and neurofilament-M (Chemicon) antibodies for 1 h at room temperature. After washing three times with PBS, the glass slides were exposed to AlexaFluor 488-labeled and AlexaFluor 596-labeled secondary antibodies (Molecular Probes, Inc., Eugene, OR) for 1 h, and then the nuclei were stained with DAPI. The coverslips were mounted in anti-fade solution (Molecular Probes). Images of the samples were captured on a fluorescence microscope. In each specimen, staining without the primary antibody was performed in a side-by-side parallel specimen as a negative control, and all controls yielded a blank image.

Statistical analysis

A total of 36 SD rats were divided into 6 groups and there were 6 rats per group. All data were expressed as the mean±SD. Statistical significance was evaluated by one-way analysis of variance (ANOVA), and multiple comparisons were performed using Scheffe’s method. A P value of less than 0.05 was considered statistically significant.

Results

LLLT at both 3 and 8 J/cm2 improved SFI in the sciatic nerve-crushed rats

To examine the functional recovery in rats with a sciatic nerve crush, we detected the SFI and ROM using gait analysis. The sham-operated rat left hindlimb was able to raise the heel and open the toes (Fig. 1A), and the SFI was approximately ?10%. After the crush injury to the rat’s sciatic nerve, the hindlimb was unable to raise the heel and open the toes (Fig. 1B), and the SFI decreased to ?120% (Fig. 1C). These results demonstrated effective sciatic nerve injury leading to declined locomotion. After 20 days, we found significant improvement of the SFI in the groups receiving 3 and 8 J/cm2 LLLT compared with the crush group without irradiation. However, we did not observe significant SFI improvement in the 15 J/cm2 LLLT group (Fig. 1C).

Figure 1

The 3 J/cm2 and 8 J/cm2 LLLT treatments improved the sciatic functional index (SFI) in rats with a sciatic nerve crush injury.

LLLT at 8 J/cm2 improved ROM in the sciatic nerve-crushed rats

Fig. 2A shows that the sham-operated left rat hindlimb had a large ROM angle when the foot was at the mid-stance phase whereas the sciatic-injured right rat hindlimb had a small ROM angle when the foot was at the mid-stance phase (Fig. 2B). The ROM of each hindlimb was measured during the mid-stance phase on both sides, and the ratio of the injured (right hindlimb) to the intact (right hindlimb) side was calculated (Fig. 2C). As shown in Fig. 2C, a significant improvement in the ROM was found in rats receiving 8 J/cm2 LLLT after the sciatic nerve crush injury. With lower (3 J/cm2) or higher (15 J/cm2) doses, no further improvement was found.

Figure 2

The 8 J/cm2 dosage of 808-nm laser enhanced the range of motion (ROM).

LLLT enhanced the myelin sheath thickness in the sciatic nerve-crushed rats

Regeneration after axonotmesis occurs with reactive Schwann cells and preserved endoneurium. We investigated the sciatic nerve regeneration using TEM analysis to evaluate the myelin sheath thickness. As shown in Fig. 3A, the TEM micrographs revealed a dense myelin sheath around the normal sciatic nerve. The integrity of the myelin sheath was obviously disrupted after the crush injury. The myelin sheath thickness analysis showed that the sciatic nerve of the normal group had a thicker myelin sheath than that of the crush group. In the sciatic nerve-crushed groups, the groups treated with LLLT presented significantly thicker myelin sheaths than the untreated crush group (Fig. 3B).

Figure 3

LLLT-treated (808 nm) sciatic nerve presented thicker myelin sheaths.

LLLT enhanced GAP43 expression in the sciatic nerve-crushed rats

In the sciatic nerve-crushed rats that received 3, 8 and 15 J/cm2 LLLT, the expression of the regeneration marker GAP43 was determined using western blotting (Fig. 4) and immunofluorescence analysis (Fig. 5 and Fig. 6) at the laser-treated site and a distal site of the sciatic nerve. The results showed that in normal rats with 8 J/cm2 LLLT, the GAP43 protein was not expressed at the sciatic nerve of the left normal hindlimb (normal) or the sciatic nerve of right hindlimb at the laser-treated (Fig. 4 and Fig. 5) or distal site (Fig. 4 and Fig. 6). In the sciatic nerve-crushed groups, the GAP43 protein levels were significantly higher after LLLT compared with groups without laser irradiation. In the laser-treated site, the 3 J/cm2-treated group showed higher levels of GAP43 expression than the 8 J/cm2-treated group (Fig. 4 and Fig. 5), whereas there was no additional effect on the left normal sciatic nerve control group (Fig. 4). We also found that in the nerve distal to the crush site, the 8 J/cm2-treated group showed even higher GAP43 expression levels than the 3 J/cm2 and 15 J/cm2 groups (Fig. 4 and Fig. 6).

Figure 4

Detection of GAP43 protein expression in rats with sciatic nerve crush injury after 808-nm LLLT treatments using western blot analysis.
Figure 5

Detection of GAP43 expression in the sciatic nerve of 808-nm LLLT laser-treated site using immunofluorescent staining.
Figure 6

Detection of GAP43 expression in the sciatic nerve of 808-nm LLLT laser-treated distal site using immunofluorescent staining.

Discussion

The present study evaluated the effects of low-level laser irradiation on peripheral nerve regeneration following rat sciatic nerve crush injury. The results indicated that the application of 808-nm LLLT at 3 and 8 J/cm2 for 20 days is effective in the promotion of sciatic nerve regeneration, as determined by molecular, morphologic and functional assessments.

Peripheral nerve injuries lead to Wallerian degeneration distal to the lesion, involving axonal loss and myelin sheath degradation, and retrograde degeneration proximal to the lesion, involving nerve cell body degradation [3]. The sciatic nerve is responsible for the motor control and sensory innervation of the lower limbs. Injury to the sciatic nerve could result in secondary muscular atrophy, resulting in varying degrees of disabilities. Although self-repair of the peripheral nerve is possible, the regeneration is slow. Microsurgical approaches have been advanced [4], [5]; nevertheless, non-surgical approaches such as physical modalities have also been proposed to facilitate nerve regeneration [6], [8], [9].

LLLT has been widely applied in clinical practice for the facilitation of nerve regeneration. Studies have shown that Schwann cells, the principal glial cells of the peripheral nervous system, secrete neurotrophic factors that promote the regeneration of the peripheral nerve [29], [30]. Phototherapy can stimulate Schwann cell proliferation in vitro [31]. In vivo studies [20][23], [32] assessing functional recovery, histological and micro-morphological changes and electrophysiological improvement after the introduction of LLLT proved to have beneficial effects on the regeneration of rat sciatic nerve injury. LLLT also accelerated the repair of transected sciatic nerves in addition to nerve conduits for gapped peripheral nerves [32], [33]. Moges et al. reported that light therapy for 14 consecutive days using 810 nm LLLT at energy density of 25 J/cm2 enhanced nerve regeneration and accelerated functional recovery after autologous nerve graft repair using fibrin glue in a rat model of median nerve transection injury [32]. The irradiation parameters described were different from our present study, in which we showed that light therapy for 20 consecutive days using 808-nm LLLT at a low energy density (3 J/cm2 and 8 J/cm2) accelerated functional recovery and enhanced nerve regeneration in sciatic nerve crush rat injury model.

Although various assessments are used with regard to peripheral nerve regeneration, functional recovery is thought to be the primary consideration that links most directly to improved functional performance after peripheral nerve injury. The SFI proposed by Bain et al is one of the most commonly used parameters to assess the functional recovery of damaged rat sciatic nerves [28]. In the study of Shen et al, correlating SFI to muscle strength, electrophysiological and morphological assessments after peripheral nerve injury or repair, indicated that the SFI is a reliable index for evaluating rat sciatic nerve regeneration [34]. However, the reliability of the SFI should be carefully assessed when using it as a single parameter to evaluate more severe sciatic nerve injuries, such as transected sciatic nerve with gaps or longer segment of crush injury. Shenaq et al assessed the correlation of SFI to histological results and clinical observations in rat sciatic nerve regeneration with a 1-cm gap and claimed no definite correlation in all groups over various periods postoperatively [35]. Another study by Monte-Raso et al evaluated the reproducibility of the SFI in rats with severe crush injury using a 5000-g static load on a 5-mm segment of the sciatic nerve [36]. They concluded that the SFI parameter is only reliable beginning at three weeks after a severe lesion of the sciatic nerve. In studies of Varejão et al [37] and Luís et al [38], a non-serrated clamp exerting a force of 54 N was used to create a sciatic nerve crush injury. In addition to functional assessment of reinnervation with the SFI, extensor postural thrust (EPT), withdrawal reflex latency (WRL) and kinematic analysis, morphological assessments such as myelin thickness and axon diameter were also evaluated. They recommended the combined use of various methods for a more thorough assessment of sciatic nerve regeneration and functional recovery.

Another functional evaluation is the clinical observation of gait patterns and the measurement of ankle kinematics. The ankle angle is representative of plantarflexion and dorsiflexion during the stance phase and is an important parameter in assessing the gait pattern [39]. Lin et al developed a motor functional index, the ankle stance angle, to assess rat sciatic nerve regeneration, which revealed a significant correlation with ankle joint passive ROM, and suggested an adequate reliability in the evaluation of functional recovery [40]. Computerized gait analysis also emerged in the application of assessing the recovery of hindlimb locomotion [37], [41]. In our study, we calculated the ratio of injured to non-injured ankle joint ROM and observed a significant decrease in ankle joint ROM ratio after crush injury. We also observed a significant improvement of ankle joint ROM ratio after 8 J/cm2 LLLT, which was compatible with previous study results claiming the effective use of LLLT in the promotion of sciatic nerve regeneration.

The present study assessed morphological changes by TEM and observed thicker myelin sheaths with more dense alignments in the 3 and 8 J/cm2 irradiated groups. A previous study by Mohammed et al [42] applied low-level laser irradiation to New Zealand adult rabbits with complete transection of the peroneal nerve and observed significant structural changes, such as thicker nerve fibers, more regular myelin layers and clearer nodes of Ranvier with the absence of a short node after laser treatment. Bae et al assessed the morphological changes of rat sciatic nerves after low-level laser irradiation using both light and electron microscopy and reported increased numbers of myelinated axons and decreased numbers of degenerated axons in the irradiated group compared with the control group without irradiation, which were proportional to the length of the laser treatment [22]. In addition to changes in myelination, increased numbers of Schwann cells were also found after LLLT [23], [31], suggesting the promotion of peripheral nerve regeneration through the secretion of various neurotrophic factors by Schwann cells.

GAP43 is expressed at high levels in nerve growth cones during development and is present in regenerating peripheral nerves [43], [44]. Shin et al found elevated GAP43 immunoreactivity in regenerating peripheral nerves after LLLT. The elevated GAP43 immunoreactivity peaked at 3 weeks after injury in the irradiated group and declined at 5 weeks in both the irradiated and non-irradiated groups, with no significant differences between the 2 groups. These findings emphasize the effect of LLLT on the early stages of the nerve recovery following sciatic nerve injury [24]. Thus, LLLT should be introduced immediately after the nerve injury. Our present study applied 20 consecutive days of low level laser irradiation immediately after a rat sciatic nerve crush injury, and the results showed increased levels of GAP43 expression in the laser-treated groups. The results were consistent with previous studies. We also analyzed the GAP43 expression level at 1 cm distal to the injured sciatic nerve, which suggested significantly higher levels of expression in the group treated with 8 J/cm2 laser irradiation. LLLT at 8 J/cm2 may have had a stronger influence on the injured sciatic nerve tissue by promoting increased regeneration efficiency and extending GAP43 expression to sites distal to the crushed region.

Varying effects of LLLT have been reported; negative effects of laser therapy have been reported by Chen et al [45]. A 904-nm GaAs laser source with pulsed laser irradiation was used over the rat sciatic nerve with a 10-mm gap, beginning a week after nerve repair. The results showed a significantly lower percentage of successful regeneration, a less mature structural organization with a smaller cross-sectional area and a lower number of myelinated axons in the laser-treated groups compared with the control group. Bagis et al also claimed no effect of a GaAs laser (904 nm) on the intact skin of the injured rat sciatic nerve, based on a lack of significant differences in the action potential parameters and histological assessments [46]. Carla et al demonstrated that no differences in recovery were observed in animals with sciatic nerve injury receiving 808-nm LLLT at 10 or 50 J/cm2 [47]. The irradiation parameters described were different from our present study, in which we showed that an 808-nm GaAlAs near-infrared laser applied with continuous irradiation to a fluence of 3 J/cm2 and 8 J/cm2 to an injured rat sciatic nerve immediately after the crush injury was able to provide functional gait recovery and led to increases in myelin sheath thickness and the neuronal growth marker GAP43. The most effective laser application, with regard to optimal wavelength, energy density and pulsed or continuous wave, is still controversial, but an early introduction of LLLT is suggested to facilitate nerve regeneration [24].

In the present study, we determined the immediate effect of LLLT after sciatic nerve injury using 20 consecutive days of irradiation on the functional recovery and nerve regeneration in sciatic nerve crush rat. The optimal treatment duration and long-term effects of LLLT will be evaluated in the future. Further investigations on the differences in proteomic expression in sciatic nerve after receiving LLLT will be conduct to clarify the signaling pathways of the biological effects, including controlling activation of the GAP43 gene, induced by LLLT.

Conclusion

Based on the results of the present study, we demonstrate that the application of a laser source with an 808-nm wavelength at doses of 3 and 8 J/cm2 to an injured rat sciatic nerve immediately after crush has beneficial effects on sciatic nerve regeneration, including better functional recovery and morphological changes and the increased expression of the neuronal growth marker GAP43.

Acknowledgments

We would like to thank everyone who provided us with reagents.

Funding Statement

This work was supported by grants from the National Health Research Institutes (NHRI-EX 102-9914EC), the National Science Council (101-2314-B-037-002-MY3) and the Kaohsiung Medical University Research Foundation (KMU-Q98007), Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

References

1. Navarro X, Vivo M, Valero-Cabre A (2007) Neural plasticity after peripheral nerve injury and regeneration. Prog Neurobiol 82: 163–201. [PubMed]
2. Allodi I, Udina E, Navarro X (2012) Specificity of peripheral nerve regeneration: interactions at the axon level. Prog Neurobiol 98: 16–37. [PubMed]
3. Navarro X, Vivó M, Valero-Cabré A (2007) Neural plasticity after peripheral nerve injury and regeneration. Progress in Neurobiology 82: 163–201. [PubMed]
4. Colen KL Choi M, Chiu DT (2009) Nerve grafts and conduits. Plast Reconstr Surg 124: e386–394. [PubMed]
5. Gu X, Ding F, Yang Y, Liu J (2011) Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol 93: 204–230. [PubMed]
6. Al-Majed AA, Neumann CM, Brushart TM, Gordon T (2000) Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci 20: 2602–2608. [PubMed]
7. Geremia NM, Gordon T, Brushart TM, Al-Majed AA, Verge VM (2007) Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression. Exp Neurol 205: 347–359. [PubMed]
8. Gordon T, Udina E, Verge VM, de Chaves EI (2009) Brief electrical stimulation accelerates axon regeneration in the peripheral nervous system and promotes sensory axon regeneration in the central nervous system. Motor Control13: 412–441. [PubMed]
9. Bannaga A, Guo T, Ouyang X, Hu D, Lin C, et al. (2002) Magnetic stimulation accelerating rehabilitation of peripheral nerve injury. J Huazhong Univ Sci Technolog Med Sci 22: 135–139. [PubMed]
10. Kokai LE, Bourbeau D, Weber D, McAtee J, Marra KG (2011) Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair. Tissue Eng Part A 17: 1263–1275. [PMC free article] [PubMed]
11. Erba P, Mantovani C, Kalbermatten DF, Pierer G, Terenghi G, et al. (2010) Regeneration potential and survival of transplanted undifferentiated adipose tissue-derived stem cells in peripheral nerve conduits. J Plast Reconstr Aesthet Surg 63: 20. [PubMed]
12. Chow RT, Johnson M, Lopes-Martins RA, Bjordal JM (2009) Efficacy of low-level laser therapy in the management of neck pain: a systematic review and meta-analysis of randomised placebo or active-treatment controlled trials. Lancet374: 1897–1908. [PubMed]
13. Brosseau L, Robinson V, Wells G, Debie R, Gam A, et al. (2005) Low level laser therapy (Classes I, II and III) for treating rheumatoid arthritis. Cochrane Database Syst Rev 19: CD002049. [PubMed]
14. Tumilty S, Munn J, McDonough S, Hurley DA, Basford JR, et al. (2010) Low level laser treatment of tendinopathy: a systematic review with meta-analysis. Photomed Laser Surg 28: 3–16. [PubMed]
15. Güngörmü? M, Akyol U (2009) The effect of gallium-aluminum-arsenide 808-nm low-level laser therapy on healing of skin incisions made using a diode laser. Photomed Laser Surg 27: 895–899. [PubMed]
16. Mohammed IF, Al-Mustawfi N, Kaka LN (2007) Promotion of regenerative processes in injured peripheral nerve induced by low-level laser therapy. Photomed Laser Surg 25: 107–111. [PubMed]
17. Gigo-Benato D, Geuna S, Rochkind S (2005) Phototherapy for enhancing peripheral nerve repair: A review of the literature. Muscle & Nerve 31: 694–701.[PubMed]
18. Rochkind S, Drory V, Alon M, Nissan M, Ouaknine GE (2007) Laser phototherapy (780 nm), a new modality in treatment of long-term incomplete peripheral nerve injury: a randomized double-blind placebo-controlled study. Photomed Laser Surg 25: 436–442. [PubMed]
19. Gigo-Benato D, Geuna S, de Castro Rodrigues A, Tos P, Fornaro M, et al. (2004) Low-power laser biostimulation enhances nerve repair after end-to-side neurorrhaphy: a double-blind randomized study in the rat median nerve model. Lasers in Medical Science 19: 57–65. [PubMed]
20. Gigo-Benato D, Russo TL, Tanaka EH, Assis L, Salvini TF, et al. (2010) Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers Surg Med 42: 673–682. [PubMed]
21. Barbosa RI, Marcolino AM, de Jesus Guirro RR, Mazzer N, Barbieri CH, et al. (2010) Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion. Lasers Med Sci 25: 423–430.[PubMed]
22. Bae CS, Lim S, Kim KY, Song CH, Pak S, et al. (2004) Effect of Ga-as laser on the regeneration of injured sciatic nerves in the rat. In Vivo 18: 489–495.[PubMed]
23. Câmara CN, Brito MV, Silveira EL, Silva DS, Simões VR, et al. (2011) Histological analysis of low-intensity laser therapy effects in peripheral nerve regeneration in Wistar rats. Acta Cir Bras 26: 12–18. [PubMed]
24. Shin DH, Lee E, Hyun JK, Lee SJ, Chang YP, et al. (2003) Growth-associated protein-43 is elevated in the injured rat sciatic nerve after low power laser irradiation. Neurosci Lett 344: 71–74. [PubMed]
25. Kupers R, Yu W, Persson JK, Xu XJ, Wiesenfeld-Hallin Z (1998) Photochemically-induced ischemia of the rat sciatic nerve produces a dose-dependent and highly reproducible mechanical, heat and cold allodynia, and signs of spontaneous pain. Pain 76: 45–59. [PubMed]
26. Yu W, Kauppila T, Hultenby K, Persson JK, Xu XJ, et al. (2000) Photochemically-induced ischemic injury of the rat sciatic nerve: a light- and electron microscopic study. J Peripher Nerv Syst 5: 209–217. [PubMed]
27. de Medinaceli L, Freed WJ, Wyatt RJ (1982) An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp Neurol 77: 634–643. [PubMed]
28. Bain JR, Mackinnon SE, Hunter DA (1989) Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg 83: 129–138. [PubMed]
29. Gravvanis AI, Lavdas AA, Papalois A, Tsoutsos DA, Matsas R (2007) The beneficial effect of genetically engineered Schwann cells with enhanced motility in peripheral nerve regeneration: review. Acta Neurochir Suppl 100: 51–56.[PubMed]
30. Madduri S, Gander B (2010) Schwann cell delivery of neurotrophic factors for peripheral nerve regeneration. J Peripher Nerv Syst 15: 93–103. [PubMed]
31. Van Breugel HH, Bar PR (1993) He-Ne laser irradiation affects proliferation of cultured rat Schwann cells in a dose-dependent manner. J Neurocytol 22: 185–190. [PubMed]
32. Moges H, Wu X, McCoy J, Vasconcelos OM, Bryant H, et al. Effect of 810 nm light on nerve regeneration after autograft repair of severely injured rat median nerve. Lasers Surg Med 43: 901–906. [PubMed]
33. Shen CC, Yang YC, Huang TB, Chan SC, Liu BS (2013) Low-Level Laser-Accelerated Peripheral Nerve Regeneration within a Reinforced Nerve Conduit across a Large Gap of the Transected Sciatic Nerve in Rats. Evidence-based complementary and alternative medicine : eCAM 2013: 175629. [PMC free article] [PubMed]
34. Shen N, Zhu J (1995) Application of sciatic functional index in nerve functional assessment. Microsurgery 16: 552–555. [PubMed]
35. Shenaq JM, Shenaq SM, Spira M (1989) Reliability of sciatic function index in assessing nerve regeneration across a 1 cm gap. Microsurgery 10: 214–219.[PubMed]
36. Monte-Raso VV, Barbieri CH, Mazzer N, Yamasita AC, Barbieri G (2008) Is the Sciatic Functional Index always reliable and reproducible? J Neurosci Methods 170: 255–261. [PubMed]
37. Varejão AS, Cabrita AM, Meek MF, Bulas-Cruz J, Melo-Pinto P, et al. (2004) Functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp. J Neurotrauma 21: 1652–1670. [PubMed]
38. Luís AL, Amado S, Geuna S, Rodrigues JM, Simões MJ, et al. (2007) Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp. J Neurosci Methods 163: 92–104.[PubMed]
39. Varejão AS, Cabrita AM, Meek MF, Bulas-Cruz J, Gabriel RC, et al. (2002) Motion of the foot and ankle during the stance phase in rats. Muscle Nerve 26: 630–635. [PubMed]
40. Lin FM, Pan YC, Hom C, Sabbahi M, Shenaq S (1996) Ankle stance angle: a functional index for the evaluation of sciatic nerve recovery after complete transection. J Reconstr Microsurg 12: 173–177. [PubMed]
41. Varejão AS, Meio-Pinto P, Meek MF, Filipe VM, Bulas-Cruz J (2004) Methods for the experimental functional assessment of rat sciatic nerve regeneration. Neurol Res 26: 186–194. [PubMed]
42. Mohammed IF, Al-Mustawfi N, Kaka LN (2007) Promotion of Regenerative Processes in Injured Peripheral Nerve Induced by Low-Level Laser Therapy. Photomedicine and Laser Surgery 25: 107–111. [PubMed]
43. Meiri KF, Pfenninger KH, Willard MB (1986) Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones. Proc Natl Acad Sci U S A 83: 3537–3541. [PMC free article] [PubMed]
44. Xu QG, Midha R, Martinez JA, Guo GF, Zochodne DW (2008) Facilitated sprouting in a peripheral nerve injury. Neuroscience 152: 877–887. [PubMed]
45. Chen YS, Hsu SF, Chiu CW, Lin JG, Chen CT, et al. (2005) Effect of low-power pulsed laser on peripheral nerve regeneration in rats. Microsurgery 25: 83–89. [PubMed]
46. Bagis S, Comelekoglu U, Coskun B, Milcan A, Buyukakilli B, et al. (2003) No effect of GA-AS (904 nm) laser irradiation on the intact skin of the injured rat sciatic nerve. Lasers in Medical Science 18: 83–88. [PubMed]
47. Medalha CC, Di Gangi GC, Barbosa CB, Fernandes M, Aguiar O, et al. (2012) Low-level laser therapy improves repair following complete resection of the sciatic nerve in rats. Lasers Med Sci 27: 629–635. [PubMed]
Neural Regen Res. 2014 Jun 15; 9(12): 1180–1182.
doi:  10.4103/1673-5374.135323
PMCID: PMC4146286

Roles of reinforced nerve conduits and low-level laser phototherapy for long gap peripheral nerve repair

Bai-Shuan Liu, Ph.D.,1 Tsung-Bin Huang,2 and Shiuh-Chuan Chan3
1Department of Medical Imaging and Radiological Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan, China
2Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan, China
3Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan, China
Corresponding author: Bai-Shuan Liu, Department of Medical Imaging and Radiological Sciences, Central Taiwan University of Science and Technology, No. 666, Buzih Road, Beitun District, Taichung 406, Taiwan, China, wt.ude.tsutc@uilsb.
Author information ? Article notes ? Copyright and License information ?
Accepted 2014 May 28.

Peripheral nerve injuries are common in clinical practice because of traumas such as crushing and sectioning. Lesions of the nerve structure result in lost or diminished sensitivity and/or motor activity in the innervated territory. The degree of lesion depends on the specific nerve involved, the magnitude and type of pressure exerted, and the duration of the compression. The results of such injuries commonly include axonal degeneration and retrograde degeneration of the corresponding neurons in the spinal medulla, followed by very slow regeneration (Rochkind et al., 2001). The adverse effects on the daily activities of patients with a peripheral nerve injury are a determining factor in establishing the goals of early recovery (Rodriguez et al., 2004). The most severe form of nerve damage involves complete transection of the nerve, which results in the loss of sensory and motor function at the site of injury. Although a degree of recovery can be expected in most untreated nerve injuries, the process is slow and often incomplete. Moreover, despite considerable advances in microsurgical techniques, the functional results of peripheral nerve repair remain largely unsatisfactory. The regrowth of nerves across large gaps is particularly challenging, usually requiring a nerve graft to correctly bridge the proximal and distal nerve stumps. At present, nerve autografting is the most common treatment used to repair peripheral nerve defects. However, this recognized “gold standard” technique has a number of inherent disadvantages, such as limited availability of donor tissue (IJkema-Paassen et al., 2004), secondary deformities, potential differences in tissue structure and size (Nichols et al., 2004), and numbness at donor sites (Bini et al., 2004). Although xenografts and allografts have been proposed as alternatives to autografts, the success rate of these techniques remains poor, often resulting in immune rejection. Thus, researchers have invested considerable effort in developing synthetic nerve conduits for the repair of peripheral nerve defects.

Nerve guide conduit

Scientists have developed various non-degradable (Chen et al., 2000) and biodegradable (Wang et al., 2001; Bini et al., 2004; Liu, 2008; Hsu et al., 2011) materials as synthetic nerve conduits, for example, PLGA (Bini et al., 2004) and PLA (Hsu et al., 2011). Of these materials, doctors have widely used non-degradable materials such as silicone rubber in general clinical cases because of its inert and mechanical properties. However, the main disadvantages of non-degradable conduits are that they remain in situ as foreign bodies following nerve regeneration and may require removal via a second surgery, which could possibly cause damage to the nerve. In contrast, biodegradable materials potentially avoid these problems. Therefore, biodegradable conduits seem a more promising alternative for reconstructing nerve gaps. Nerve guidance channels fabricated out of collagen have already shown rather favorable results in nerve repair (Itoh et al., 2002). However, clinical experience with collagen products has demonstrated that cracks and tears can occur when the suture needle penetrates the conduits. In addition, biodegradable conduits that degrade with time may lose their functionality as a structural cuff. Accordingly, an ideal biodegradable conduit should maintain its structural integrity during the regenerative processes (Yannas and Hill, 2004).

Gelatin is less expensive and much easier to obtain in concentrated solutions than collagen. Moreover, gelatin is a biodegradable polymer with excellent biocompatibility, plasticity, and adhesiveness. However, swelling of the degradable tube walls caused by absorption of body fluids may occur during the nerve regenerative processes. This swelling could occlude the lumen and therefore impair axonal regeneration. In addition, the handling characteristics are unsatisfactory for suturing, and the lumen of gelatin channels may collapse or be obliterated following implantation. Therefore, the use of proper cross-linking agents to modulate the mechanico-chemical characteristics of gelatin is desirable in order to prevent toxicity and generate stable materials for biomedical applications. Various cross-linkers, such as formaldehyde, glutaraldehyde (Chen et al., 2005), genipin (Yang et al., 2010, 2011), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Chang et al., 2007) have been used to compensate for the disadvantages inherent in gelatin, and to make gelatin nerve substitutes resistant to natural biodegradation following transplantation. Since cross-linked gelatin may have low mechanical strength under physiological conditions, its applications may prove limited.

Previous studies (Yang et al., 2010, 2011) developed a biodegradable composite (GGT conduit) consisting of genipin cross-linked gelatin annexed with ?-tricalcium phosphate (?-TCP) ceramic particles to enhance the mechanical strength as a nerve guidance channel-material for axon regeneration (Figure 1). The results of that study revealed that the TCP ceramic particles provided structural reinforcement to the genipin-cross-linked gelatin (GG) structure. Macroscopic observations show that this study does not observe any unsatisfactory swelling or deformation of the GGT nerve guide conduits. The improvement in the water uptake and swelling ratios may have been attributable to the presence of TCP ceramic particles in the GG matrix. Consequently, the GGT conduits swelled slowly and maintained a lower water uptake and swelling ratios than the GG conduits. Therefore, the hydrated GGT conduits (when grafted in vivo to repair nerve defects) did not stenose and collapse to compress the regenerating nerve fiber of the lumen. Mechanical measurements showed that these good mechanical properties, which benefited from the addition of TCP ceramic particles, rendered it possible for the GGT conduit to resist the muscular contraction and keep its cylindrical shape unchanged within a considerable period after implantation into the body. Since the collapse of an unfilled circular conduit is a major block to nerve regeneration in tubulization, the properties of a gelatin tube that can be molded into various configurations and compounded with TCP, can effectively enhance nerve regeneration. Besides, as tricalcium phosphate dissolves during the degradation of GGT, calcium ions could be released from the conduits, and a previous study (Kulbatski et al., 2004) has shown that a post-neuritotomy rise in calcium influx through calcium channels is a necessity for neurite regeneration. In addition, the GGT conduit had the strength necessary to withstand the muscular forces that surrounded it, meaning that a stable support structure for the extended regeneration processes was maintained. These results demonstrate the feasibility of designed GGT conduits in the applications of peripheral nerve repair.

Figure 1

Smacrograph and scanning electron micrograph (SEM) of the genipin-crosslinked gelatin annexed with tricalcium phosphate (GGT) conduit.

Laser therapy

Clinicians have focused on developing more effective methods to promote nerve regeneration, target organ reinnervation, and restore function at the site of injury. Many physical and neurotrophic factors, as well as pharmaceutical drugs, influence nerve regeneration. Physiotherapy commonly involves the use of therapeutic instruments for regenerative purposes (Gigo-Benato et al., 2005). Various forms of external stimulation have been employed to accelerate the process of regeneration, which in turn accelerates functional recovery. Such techniques include electrical (Mendonça et al., 2003), ultrasound (Raso et al., 2005), and low-level laser (LLL) stimuli. Clinical and experimental studies have provided evidence that lasers can increase nerve function, reduce the formation of wounds, increase the metabolic activity of neurons, and enhance myelin production (Bagis et al., 2002). The non-invasive nature of laser phototherapy enables treatment without surgical intervention. LLL therapy began to be used in the regeneration and functional recuperation process of peripheral nerves in the 1970s, and the results obtained so far have been inconsistent. Many animal experiments and clinical studies have indicated that LLL irradiation can attenuate injury, promote repair, and stimulate axonal sprouting and propagation, but its mechanism of action is not well understood (Amat et al., 2006). A review of the literature on phototherapy for peripheral nerve repair found that the use of laser was based on several wavelengths (632–904 nm) (Masoumipoor et al., 2014), lesion types (crushing, neurorrhaphy, and tubulation), sample types, the duration and manner of the emission (Marcolino et al., 2013; Akgul et al., 2014), and the assessment types (such as functional, electrophysiological, and morphometric) (Gigo-Benato et al., 2005).

In many studies, descriptions of the irradiation parameters, such as dose, average power, time, and application methods, have expressly varied, hampering the methodological comprehension required for the reproduction of results and hindering comparisons between studies. Barbosa et al. (2010) sought to analyze the effects of two different GaAlAs laser wavelengths (660 nm and 830 nm) on sciatic nerve regeneration by using the same crushing injuries for a novel comparison of studies reported in the literature. They observed that the 660 nm wavelength treatment group had the best SFI scores on average, indicating that the use of these parameters was more efficient. The possibility that neural tissue is located in more superficial layers may have favored a better response to the shorter wavelength. Data also suggested that 660 nm LLL therapy with low (10 J/cm2) or moderate (60 J/cm2) energy densities is able to accelerate neuromuscular recovery after nerve crush injury in rats (Gigo-Benato et al., 2010). Our own previous studies investigated the influence of large-area irradiation using an aluminum-gallium-indium phosphide (AlGaInP) diode laser (660 nm) (Shen et al., 2011) and trigger point therapy using gallium-aluminum-arsenide phosphide (GaAlAsP) laser diodes (660 nm) (Shen et al., 2013a, 2013b) on the neurorehabilitation of transected sciatic nerves in rats after bridging them with the GGT nerve conduit (Figure 2). The results for these studies indicated that the GGT/laser system may be very helpful for long-gap nerve regeneration as well as for acceleration of the reinnervation rate of regenerated nerves, which may lead to sufficient morphologic and functional recovery of the peripheral nerve.

Figure 2

Transected nerve was subjected to a large-area irradiated therapy with the 660-nm aluminum-gallium-indium phosphide (AlGaInP) low-level laser (A) or a transcutaneous trigger point therapy with the 660-nm gallium aluminum arsenide phosphide (GaAlAsP) low-level

It has also previously been shown that LLL enhances Schwann cell proliferation in vitro. Schwann cells myelinate axons of the peripheral nervous system and play a crucial role in post-injury nerve regeneration. They promote neuronal survival, guide axons to their proper targets, and secrete neurotrophic factors that aid axonal elongation (Bhatheja and Field, 2006). Morphological changes in the mitochondria of lymphocytes, as well as in the proliferation of mononuclear cells, have also been observed after radiation with a red laser, and these responses might be beneficial in the process of tissue repair (Gulsoy et al., 2006). The underlying mechanism of phototherapy in nerve regeneration has been proposed in previous in vitro studies which showed that phototherapy induced Schwann cell proliferation, as well as massive neurite sprouting and outgrowth in cultured neuronal cells. It has also been suggested that phototherapy may enhance the recovery of neurons by altering the oxidative metabolism of mitochondria (Elles et al., 2003). The same mechanism may guide neuronal growth cones in vitro, perhaps through interaction with cytoplasmic proteins and, in particular, by enhancing actin polymerization at the leading edge of the axon (Ehrlicher et al., 2002). One possible molecular explanation is the increase in growth-associated protein-43 (GAP-43) immunoreactivity during the early stages of nerve regeneration proceeding phototherapy (Shin et al., 2003). In summary, all of the aforementioned effects may play a role in accelerating axonal regeneration and preventing the loss of neurons.

Although the preliminary results support the mechanical strength and biocompatibility of the GGT conduit and are encouraging in regards to peripheral nerve regeneration, further studies should attempt to improve the design of GGT nerve guide conduits. Examples of such studies could include an introduction of neurotrophic factors or seeding cells to establish the possibility of using GGT grafts as a suitable alternative to nerve autografts for peripheral nerve regeneration. With regard to clinical applicability, LLL phototherapy makes an important contribution towards the development of a safe and effective strategy for rehabilitating peripheral nerve injuries. Further studies on the use of LLL therapy as a noninvasive treatment modality for various nerve diseases and injuries could pave the way for mainstream acceptance and standardization of this innovative therapy.

References

1. Akgul T, Gulsoy M, Gulcur HO. Effects of early and delayed laser application on nerve regeneration. Lasers Med Sci. 2014;29:351–357. [PubMed]
2. Amat A, Rigau J, Waynant RW, Ilev IK, Anders JJ. The electric field induced by light can explain cellular responses to electromagnetic energy: a hypothesis of mechanism. Photochem Photobiol. 2006;82:152–160. [PubMed]
3. Bagis S, Comelekoglu U, Sahin G, Buyukakilli B, Erdogan C, Kanik A. Acute electrophysiologic effect of pulsed galliumarsenide low energy laser irradiation on configuration of compound nerve action potential and nerve excitability. Lasers Surg Med. 2002;30:376–380. [PubMed]
4. Barbosa RI, Marcolino AM, Guirro RRD, Mazzer N, Barbieri CH, Fonseca MDR. Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion. Lasers Med Sci. 2010;25:423–430.[PubMed]
5. Bhatheja K, Field J. Schwann cells: Origins and role in axonal maintenance and regeneration. Int J Biochem Cell Biol. 2006;38:1995–1999. [PubMed]
6. Bini TB, Gao S, Xu X, Wang S, Ramakrishna S, Leong KW. Peripheral nerve regeneration by microbraided poly(L-lactide-co-glycolide) biodegradable polymer fibers. J Biomed Mater Res A. 2004;68:286–295. [PubMed]
7. Chang JY, Lin JH, Yao CH, Chen JH, Lai TY, Chen YS. In vivo evaluation of a biodegradable EDC/NHS-cross-linked gelatin peripheral nerve guide conduit material. Macromol Biosci. 2007;7:500–507. [PubMed]
8. Chen PR, Chen MH, Lin FH, Su WY. Release characteristics and bioactivity of gelatin-tricalcium phosphate membranes covalently immobilized with nerve growth factors. Biomaterials. 2005;26:6579–6587. [PubMed]
9. Chen YS, Hsieh CL, Tsai CC, Chen TH, Cheng WC, Hu CL, Yao CH. Peripheral nerve regeneration using silicone rubber chambers filled with collagen, laminin and fibronectin. Biomaterials. 2000;21:1541–1547. [PubMed]
10. Ehrlicher A, Betz T, Stuhrmann B, Koch D, Milner V, Raizen MG, Kas J. Guiding neuronal growth with light. Proc Natl Acad Sci U S A. 2002;99:16024–16028. [PMC free article] [PubMed]
11. Elles JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT. Therapeutic photobiomodulation for methanol-induced retinal toxicity. Proc Natl Acad Sci U S A. 2003;100:3439–3444.[PMC free article] [PubMed]
12. Gigo-Benato D, Geuna S, Rochkind S. Phototherapy for enhancing peripheral nerve repair: a review of the literature. Muscle Nerve. 2005;31:694–701.[PubMed]
13. Gigo-Benato D, Russo TL, Tanaka EH, Assis L, Salvini TF, Parizotto NA. Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers Surg Med. 2010;42:673–682.[PubMed]
14. Gulsoy M, Ozer GH, Bozkulak O, Tabakoglu HO, Aktas E, Deniz G, Ertan C. The biological effects of 632.8-nm low energy He-Ne laser on peripheral blood mononuclear cells in vitro. J Photochem Photobiol B. 2006;82:199–202.[PubMed]
15. Hsu SH, Chan SH, Chiang CM, Chen CC, Jiang CF. Peripheral nerve regeneration using a microporous polylactic acid asymmetric conduit in a rabbit long-gap sciatic nerve transection model. Biomaterials. 2011;32:3764–3775.[PubMed]
16. IJkema-Paassen J, Jansen K, Gramsbergen A, Meek MF. Transection of peripheral nerves, bridging strategies and effect evaluation. Biomaterials. 2004;25:1583–1592. [PubMed]
17. Itoh S, Takakuda K, Kawabata S, Aso Y, Kasai K, Itoh H, Shinomiya K. Evaluation of cross-linking procedures of collagen tubes used in peripheral nerve repair. Biomaterials. 2002;23:4475–4481. [PubMed]
18. Kulbatski I, Cook DJ, Tator CH. Calcium entry through L-type calcium channels is essential for neurite regeneration in cultured sympathetic neurons. J Neurotrauma. 2004;21:357–374. [PubMed]
19. Liu BS. Fabrication and evaluation of a biodegradable proanthocy-anidin-crosslinked gelatin conduit in peripheral nerve repair. J Biomed Mater Res Part A. 2008;87:1092–1102. [PubMed]
20. Marcolino AM, Barbosa RI, das Neves LM, Mazzer N, de Jesus Guirro RR, de Cássia Registro Fonseca M. Assessment of functional recovery of sciatic nerve in rats submitted to low-level laser therapy with different fluences. An experimental study: laser in functional recovery in rats. J Hand Microsurg. 2013;5:49–53. [PMC free article] [PubMed]
21. Masoumipoor M, Jameie SB, Janzadeh A, Nasirinezhad F, Soleimani M, Kerdary M. Effects of 660- and 980-nm low-level laser therapy on neuropathic pain relief following chronic constriction injury in rat sciatic nerve. Lasers Med Sci. 2014 Epub ahead of print. [PubMed]
22. Mendonça AC, Barbieri CH, Mazzer N. Directly applied low intensity direct electric current enhances peripheral nerve regeneration in rats. J Neurosci Methods. 2003;129:183–190. [PubMed]
23. Nichols CM, Brenner MJ, Fox IK, Tung TH, Hunter DA, Rickman SR, Mackinnon SE. Effects of motor versus sensory nerve grafts on peripheral nerve regeneration. Exp Neurol. 2004;190:347–355. [PubMed]
24. Raso VVM, Barbieri CH, Mazzer N, Fasan VS. Can therapeutic ultrasound influence the regeneration of the peripheral nerves? J Neurosci Methods. 2005;142:185–192. [PubMed]
25. Rochkind S, Nissan M, Alon M, Shamir M, Salame K. Effects of laser irradiation on the spinal cord for the regeneration of crushed peripheral nerve in rats. Lasers Surg Med. 2001;28:216–219. [PubMed]
26. Rodriguez FJ, Valero-Cabré A, Navarro X. Regeneration and functional recovery following peripheral nerve injury. Drug Discov Today Dis Models. 2004;1:177–185.
27. Shen CC, Yang YC, Huang TB, Chan SC, Liu BS. Low-level laser-accelerated peripheral nerve regeneration within a reinforced nerve conduit across a large gap of the transected sciatic nerve in rats. Evid Based Complement Alternat Med. 2013a;2013:175629. [PMC free article] [PubMed]
28. Shen CC, Yang YC, Huang TB, Chan SC, Liu BS. Neural regeneration in a novel nerve conduit across a large gap of the transected sciatic nerve in rats with low-level laser phototherapy. J Biomed Mater Res A. 2013b;101:2763–2777.[PubMed]
29. Shen CC, Yang YC, Liu BS. Large-area irradiated low-level laser effect in a biodegradable nerve guide conduit on neural regeneration of peripheral nerve injury in rats. Injury. 2011;42:803–813. [PubMed]
30. Shin DH, Lee E, Hyun JK, Lee SJ, Chang YP, Kim JW, Choi YS, Kwon BS. Growth-associated protein-43 is elevated in the injured rat sciatic nerve after low power laser irradiation. Neurosci Lett. 2003;344:71–74. [PubMed]
31. Wang S, Wan ACA, Xu X, Gao S, Mao HQ, Leong KW, Yu H. A new nerve guide conduit material composed of a biodegradable poly(phosphoester) Biomaterials. 2001;22:1157–1169. [PubMed]
32. Yang YC, Shen CC, Cheng HC, Liu BS. Sciatic nerve repair by reinforced nerve conduits made of gelatin-tricalcium phosphate composites. J Biomed Mater Res A. 2011;96:288–300. [PubMed]
33. Yang YC, Shen CC, Huang TB, Chang SH, Cheng HC, Liu BS. Characteristics and biocompatibility of a biodegradable genipin-crosslinked gelatin/?-tricalcium phosphate reinforced nerve guide conduit. J Biomed Mater Res B Appl Biomater. 2010;95:207–217. [PubMed]
34. Yannas IV, Hill BJ. Selection of biomaterials for peripheral nerve regeneration using data from the nerve chamber model. Biomaterials. 2004;25:1593–1600. [PubMed]
Lasers Med Sci.  2011 Aug 11. [Epub ahead of print]

Functional and morphometric differences between the early and delayed use of phototherapy in crushed median nerves of rats.

Santos AP, Suaid CA, Xavier M, Yamane F.

Source

Department of Physiotherapy, Federal University of Jequitinhonha and Mucuri Valleys, Campus JK – Rodovia MGT 367 – Km 583, nº 5000 – Alto da Jacuba CEP: 39100-000, Diamantina, MG, Brazil, apsfisio@hotmail.com.

Abstract

This study evaluated the functional and quantitative differences between the early and delayed use of phototherapy in crushed median nerves. After a crush injury, low-level laser therapy (GaAs) was applied transcutaneously at the injury site, 3 min daily, with a frequency of five treatments per week for 2 weeks. In the early group, the first laser treatment started immediately after surgery, and in the delayed group, after 7 days. The grasping test was used for functional evaluation of the median nerve, before, 10, and 21 days after surgery, when the rats were killed. Three segments of the median nerve were analyzed histomorphometrically by light microscopy and computer analysis. The following features were observed: myelinated fiber and axon diameters, myelin sheath area, g-ratio, density and number of myelinated fibers, and area and number of capillaries. In the proximal segment (site of crush), the nerves of animals submitted to early and delayed treatment showed myelinated fiber diameter and myelin sheath area significantly larger compared to the untreated group. In the distal segment, the myelin sheath area was significantly smaller in the untreated animals compared to the delayed group. The untreated, early, and delayed groups presented a 50, 57, and 81% degree of functional recovery, respectively, at 21 days after injury, with a significant difference between the untreated and delayed groups. The results suggest that the nerves irradiated with low-power laser exhibit myelinated fibers of greater diameter and a better recovery of function.

Acta Cir Bras. 2011 Feb;26(1):12-18.

Histological analysis of low-intensity laser therapy effects in peripheral nerve regeneration in Wistar rats.

Câmara CN, Brito MV, Silveira EL, Silva DS, Simões VR, Pontes RW.

Department of Physiotherapy, UNAMA, Belem, PA, Brazil.

Abstract

Purpose: Analyze the influence of low-intensity laser therapy in the sciatic nerve regeneration of rats submitted to controlled crush through histological analysis. Methods: Were used 20 Wistar rats, to analyze the influence of low-intensity laser therapy in the sciatic nerve regeneration, where the injury of the type axonotmesis was induced by a haemostatic clamp Crile (2nd level of the rack). The animals were randomly distributed in 2 groups. Control group (CG n = 10) and Laser group (LG n = 10). These were subdivided in 2 subgroups each, according to the euthanasia period: (CG14 _ n = 5 and CG21 _ n = 5) and (LG14 _ n = 5 and LG21 _ n = 5). At the end of treatment, the samples were removed and prepared for histological analysis, where were analyzed and quantified the following findings: Schwann cells, myelinic axons with large diameter and neurons. Results: In the groups submitted to low-intensity laser therapy, were observed an increase in the number of all analyzed aspects with significance level. Conclusion: The irradiation with low intensity laser (904nm) influenced positively the regeneration of the sciatic nerve in Wistar rats after being injured by crush (axonotmesis), becoming the nerve recovery more rapid and efficient.

Lasers Med Sci. 2010 May;25(3):423-30. Epub 2010 Feb 6.

Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion.

 

Barbosa RI, Marcolino AM, de Jesus Guirro RR, Mazzer N, Barbieri CH, de Cássia Registro Fonseca M.

Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, Medical School of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto 14049-900, SP, Brazil.  ribarbosa@hcrp.fmrp.usp.br

Abstract

Peripheral nerves are structures that, when damaged, can result in significant motor and sensory disabilities. Several studies have used therapeutic resources with the aim of promoting early nerve regeneration, such as the use of low-power laser. However, this laser therapy does not represent a consensus regarding the methodology, thus yielding controversial conclusions. The objective of our study was to investigate, by functional evaluation, the comparative effects of low-power laser (660 nm and 830 nm) on sciatic nerve regeneration following crushing injuries. Twenty-seven Wistar rats subjected to sciatic nerve injury were divided into three groups: group sham, consisting of rats undergoing simulated irradiation; a group consisting of rats subjected to gallium-aluminum-arsenide (GaAlAs) laser at 660 nm (10 J/cm(2), 30 mW and 0.06 cm(2) beam), and another one consisting of rats subjected to GaAlAs laser at 830 nm (10 J/cm(2), 30 mW and 0.116 cm(2)). Laser was applied to the lesion for 21 days. A sciatic functional index (SFI) was used for functional evaluation prior to surgery and on days 7, 14, and 21 after surgery. Differences in SFI were found between group 660 nm and the other ones at the 14th day. One can observe that laser application at 660 nm with the parameters and methods utilised was effective in promoting early functional recovery, as indicated by the SFI, over the period evaluated.

BMC Complement Altern Med. 2009 Apr 15;9:8.

Low infra red laser light irradiation on cultured neural cells: effects on mitochondria and cell viability after oxidative stress.

Giuliani A, Lorenzini L, Gallamini M, Massella A, Giardino L, Calzà L.

BioPharmaNet-DIMORFIPA, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell’Emilia, Bologna, Italy. a.giuliani@unibo.it

BACKGROUND: Considerable interest has been aroused in recent years by the well-known notion that biological systems are sensitive to visible light. With clinical applications of visible radiation in the far-red to near-infrared region of the spectrum in mind, we explored the effect of coherent red light irradiation with extremely low energy transfer on a neural cell line derived from rat pheochromocytoma. We focused on the effect of pulsed light laser irradiation vis-à-vis two distinct biological effects: neurite elongation under NGF stimulus on laminin-collagen substrate and cell viability during oxidative stress.

METHODS: We used a 670 nm laser, with extremely low peak power output (3 mW/cm2) and at an extremely low dose (0.45 mJ/cm2). Neurite elongation was measured over three days in culture. The effect of coherent red light irradiation on cell reaction to oxidative stress was evaluated through live-recording of mitochondria membrane potential (MMP) using JC1 vital dye and laser-confocal microscopy, in the absence (photo bleaching) and in the presence (oxidative stress) of H2O2, and by means of the MTT cell viability assay.

RESULTS: We found that laser irradiation stimulates NGF-induced neurite elongation on a laminin-collagen coated substrate and protects PC12 cells against oxidative stress.

CONCLUSION: These data suggest that red light radiation protects the viability of cell culture in case of oxidative stress, as indicated by MMP measurement and MTT assay. It also stimulates neurite outgrowth, and this effect could also have positive implications for axonal protection.

Int Rev Neurobiol. 2009;87:445-64.

Chapter 25: Phototherapy in peripheral nerve injury: effects on muscle preservation and nerve regeneration.

Rochkind S, Geuna S, Shainberg A.

Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University, Israel.

Posttraumatic nerve repair and prevention of muscle atrophy represent a major challenge of restorative medicine. Considerable interest exists in the potential therapeutic value of laser phototherapy for restoring or temporarily preventing denervated muscle atrophy as well as enhancing regeneration of severely injured peripheral nerves. Low-power laser irradiation (laser phototherapy) was applied for treatment of rat denervated muscle in order to estimate biochemical transformation on cellular and tissue levels, as well as on rat sciatic nerve model after crush injury, direct or side-to-end anastomosis, and neurotube reconstruction. Nerve cells’ growth and axonal sprouting were investigated in embryonic rat brain cultures. The animal outcome allowed clinical double-blind, placebo-controlled randomized study that measured the effectiveness of 780-nm laser phototherapy on patients suffering from incomplete peripheral nerve injuries for 6 months up to several years. In denervated muscles, animal study suggests that the function of denervated muscles can be partially preserved by temporary prevention of denervation-induced biochemical changes. The function of denervated muscles can be restored, not completely but to a very substantial degree, by laser treatment initiated at the earliest possible stage post injury. In peripheral nerve injury, laser phototherapy has an immediate protective effect. It maintains functional activity of the injured nerve for a long period, decreases scar tissue formation at the injury site, decreases degeneration in corresponding motor neurons of the spinal cord, and significantly increases axonal growth and myelinization. In cell cultures, laser irradiation accelerates migration, nerve cell growth, and fiber sprouting. In a pilot, clinical, double-blind, placebo-controlled randomized study in patients with incomplete long-term peripheral nerve injury, 780-nm laser irradiation can progressively improve peripheral nerve function, which leads to significant functional recovery. A 780-nm laser phototherapy temporarily preserves the function of a denervated muscle, and accelerates and enhances axonal growth and regeneration after peripheral nerve injury or reconstructive procedures. Laser activation of nerve cells, their growth, and axonal sprouting can be considered as potential treatment for neural injury. Animal and clinical studies show the promoting action of phototherapy on peripheral nerve regeneration, which makes it possible to suggest that the time for broader clinical trials has come.

Lasers Surg Med. 2009 Apr;41(4):277-81.

Increase in neuronal sprouting and migration using 780 nm laser phototherapy as procedure for cell therapy.

Rochkind S, El-Ani D, Nevo Z, Shahar A.

Division of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv 64239, Israel. rochkind@zahav.net.il

BACKGROUND AND OBJECTIVES: The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR-Gel (cross-linked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell-MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated.

MATERIALS AND METHODS: Whole brains were dissected from 16 days Sprague-Dawley rat embryos. Cells were mechanically dissociated, using narrow pipettes, and seeded on positively charged cylindrical MCs. After 4-14 days in suspension, the formed floating cell-MC aggregates were seeded as stationary cultures in NVR-Gel. Single cell-MC aggregates were either irradiated with near-infrared 780 nm laser beam for 1, 4, or 7 minutes, or cultured without irradiation. Laser powers were 10, 30, 50, 110, 160, 200, and 250 mW.

RESULTS: 780 nm laser irradiation accelerated fiber sprouting and neuronal cell migration from the aggregates. Furthermore, unlike control cultures, the irradiated cultures (mainly after 1 minute irradiation of 50 mW) were already established after a short time of cultivation. They contained a much higher number of large size neurons (P<0.01), which formed dense branched interconnected networks of thick neuronal fibers.

CONCLUSIONS: 780 nm laser phototherapy of embryonic rat brain cultures embedded in hyaluronic acid-laminin gel and attached to positively charged cylindrical MCs, stimulated migration and fiber sprouting of neuronal cells aggregates, developed large size neurons with dense branched interconnected network of neuronal fibers and, therefore, can be considered as potential procedure for cell therapy of neuronal injury or disease.

Neurosurg Focus. 2009;26(2):E8

Phototherapy in peripheral nerve regeneration: From basic science to clinical study.

Rochkind S.

Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel.

Object This review summarizes the continuous study of low-power laser radiation treatment of a severely injured peripheral nerve. Laser phototherapy was applied as a supportive factor for accelerating and enhancing axonal growth and regeneration after injury or a reconstructive peripheral nerve procedure. In nerve cell cultures, laser phototherapy was used to stimulate activation of nerve cells.

Methods Low-power laser radiation was used for treatment of peripheral nerve injury using a rat sciatic nerve model after crush injury, neurorrhaphy, or neurotube reconstruction. Nerve cell growth and axonal sprouting were investigated using laser phototherapy on embryonic rat brain cultures. The outcome in animal studies facilitated a clinical double-blind, placebo-controlled, randomized study that measured the effectiveness of 780-nm laser phototherapy on patients suffering from incomplete peripheral nerve injuries for 6 months to several years.

Results Animal studies showed that laser phototherapy has an immediate protective effect, maintains functional activity of the injured nerve, decreases scar tissue formation at the injury site, decreases degeneration in corresponding motor neurons of the spinal cord, and significantly increases axonal growth and myelinization. In cell cultures, laser irradiation accelerates migration, nerve cell growth, and fiber sprouting. A pilot clinical double-blind, placebocontrolled, randomized study showed that in patients with incomplete long-term peripheral nerve injury, 780-nm laser radiation can progressively improve peripheral nerve function, which leads to significant functional recovery.

Conclusions Using 780-nm laser phototherapy accelerates and enhances axonal growth and regeneration after injury or a reconstructive peripheral nerve procedure. Laser activation of nerve cells, their growth, and axonal sprouting can be considered as potential treatment of neuronal injury. Animal and clinical studies show the promoting action of phototherapy on peripheral nerve regeneration, making it possible to suggest that the time for broader clinical trials has arrived.

Gen Dent. 2008 Nov-Dec;56(7):629-34.

Low level lasers in dentistry.

Ross G, Ross A.

Laser Light Canada.

Low level laser therapy (LLLT) uses light energy, in the form of adenosine triphosphate (ATP), to elicit biological responses in the body. The increased cellular energy and changes in the cell membrane permeability result in pain relief, wound healing, muscle relaxation, immune system modulation, and nerve regeneration. This article investigates the clinical effects of LLLT and explains how it can be applied in the dental field.

Photomed Laser Surg. 2007 Oct;25(5):436-42

Laser phototherapy (780 nm), a new modality in treatment of long-term incomplete peripheral nerve injury: a randomized double-blind placebo-controlled study.

Rochkind S, Drory V, Alon M, Nissan M, Ouaknine GE.

Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University, Israel. rochkind@zahav.net.il

OBJECTIVE: The authors conducted this pilot study to prospectively investigate the effectiveness of low-power laser irradiation (780 nm) in the treatment of patients suffering from incomplete peripheral nerve and brachial plexus injuries for 6 months up to several years.

BACKGROUND DATA: Injury of a major nerve trunk frequently results in considerable disability associated with loss of sensory and motor functions. Spontaneous recovery of long-term severe incomplete peripheral nerve injury is often unsatisfactory.

METHODS: A randomized, double-blind, placebo-controlled trial was performed on 18 patients who were randomly assigned placebo (non-active light: diffused LED lamp) or low-power laser irradiation (wavelength, 780 nm; power, 250 mW). Twenty-one consecutive daily sessions of laser or placebo irradiation were applied transcutaneously for 3 h to the injured peripheral nerve (energy density, 450 J/mm(2)) and for 2 h to the corresponding segments of the spinal cord (energy density, 300 J/mm(2)). Clinical and electrophysiological assessments were done at baseline, at the end of the 21 days of treatment, and 3 and 6 months thereafter.

RESULTS: The laser-irradiated and placebo groups were in clinically similar conditions at baseline. The analysis of motor function during the 6-month follow-up period compared to baseline showed statistically significant improvement (p = 0.0001) in the laser-treated group compared to the placebo group. No statistically significant difference was found in sensory function. Electrophysiological analysis also showed statistically significant improvement in recruitment of voluntary muscle activity in the laser-irradiated group (p = 0.006), compared to the placebo group.

CONCLUSION: This pilot study suggests that in patients with long-term peripheral nerve injury noninvasive 780-nm laser phototherapy can progressively improve nerve function, which leads to significant functional recovery.

Photomed Laser Surg. 2007 Jun;25(3):137-43

Efficacy of 780-nm laser phototherapy on peripheral nerve regeneration after neurotube reconstruction procedure (double-blind randomized study).

Rochkind S, Leider-Trejo L, Nissan M, Shamir MH, Kharenko O, Alon M.

Division of Peripheral Nerve Reconstruction, Tel-Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel. rochkind@zahav.net.il

OBJECTIVE: This pilot double-blind randomized study evaluated the efficacy of 780-nm laser phototherapy on the acceleration of axonal growth and regeneration after peripheral nerve reconstruction by polyglycolic acid (PGA) neurotube.

BACKGROUND DATA: The use of a guiding tube for the reconstruction of segmental loss of injured peripheral nerve has some advantages over the regular nerve grafting procedure. Experimental studies have shown that laser phototherapy is effective in influencing nerve regeneration.

METHODS: The right sciatic nerve was transected, and a 0.5-cm nerve segment was removed in 20 rats. A neurotube was placed between the proximal and the distal parts of the nerve for reconnection of nerve defect. Ten of 20 rats received post-operative, transcutaneous, 200-mW, 780-nm laser irradiation for 14 consecutive days to the corresponding segments of the spinal cord (15 min) and to the reconstructed nerve (15 min).

RESULTS: At 3 months after surgery, positive somato-sensory evoked responses were found in 70% of the irradiated rats (p = 0.015), compared to 30% of the non-irradiated rats. The Sciatic Functional Index in the irradiated group was higher than in the non-irradiated group (p < 0.05). Morphologically, the nerves were completely reconnected in both groups, but the laser-treated group showed an increased total number of myelinated axons.

CONCLUSION: The results of this study suggest that postoperative 780-nm laser phototherapy enhances the regenerative process of the peripheral nerve after reconnection of the nerve defect using a PGA neurotube.

Photomed Laser Surg. 2007 Apr;25(2):107-11

Promotion of regenerative processes in injured peripheral nerve induced by low-level laser therapy.

Mohammed IF, Al-Mustawfi N, Kaka LN.

Department of Anatomy, Al-Kindy Medical College, Baghdad University, Baghdad, Iraq. ihsan20042002@yahoo.com

OBJECTIVE: This study aimed to assess in vitro the influence of low-level laser therapy (LLLT) on the regenerative processes of a peripheral nerve after trauma.

BACKGROUND DATA: In peripheral nerve injury initiated after severing due to accident or by a surgeon during operation, photomodulation by light in the red to near-infrared range (530-1000 nm) using low-energy lasers has been shown to accelerate nerve regeneration.

METHOD: Twenty-four New Zealand adult male rabbits were randomly assigned to two equal groups (control and laser-treated). General anesthesia was administered intramuscularly, and exploration of the peroneal nerve was done in the lateral aspect of the left leg. Complete section of the nerve was performed, which was followed by suturing of the neural sheath (epineurium). Irradiation was carried out directly after the operation and for 10 consecutive days. The laser used was diode with wavelength of 901 nm (impulsive) and power of 10 mW; it was a square-shaped window type (16 cm(2)), and its energy was applied by direct contact of the instrument’s window to the site of the operation. Three rabbits from each group were sacrificed at the end of weeks 2, 4, 6, and 8, and specimens were collected from the site of nerve suturing and sent for histopathological examination.

RESULTS: Two important factors were examined via histopathology: diameter of the nerve fibers and individual internodal length. Compared to the control group, significant variations in regeneration were observed, including thicker nerve fibers, more regular myelin layers, clearer nodes of Ranvier with absence of short nodes in the treated group. Variations between the two groups for diameter were significant for the 2(nd) week (p < 0.05), highly significant for the 4(th) and 6(th) weeks, respectively (p < 0.01), and very highly significant for the 8(th) week (p < 0.001). Variations between the two groups for internodal length were highly significant for the 2(nd) and 4(th) weeks (p < 0.01), and very highly significant for the 6(th) and 8(th) weeks (p < 0.001).

CONCLUSION: This experiment affirms the beneficial effect of LLLT on nerve regeneration, since LLLT produced a significant amount of structural and cellular change. The results of the present study suggest that laser therapy may be a viable approach for nerve regeneration, which may be of clinical relevance in scheduled surgery or microsurgery.

Neurol Res. 2004 Mar;26(2):161-6

Further development of reconstructivev and cell tissue-engineering technology for treatment of complete peripheral nerve injury in rats.

Rochkind S, Astachov L, el-Ani D, Hayon T, Graif M, Barsky L, Alon M, Odvak I, Nevo Z, Shahar A.

Department of Neurosurgery, Division of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel. rochkind@zahav.net.il

In this work we evaluated the efficacy of biodegradable composite co-polymer guiding neurotube, based on tissue-engineering technology, for the treatment of complete peripheral nerve injury where the nerve defect is significant. The right sciatic nerve of 12 three-month-old rats was completely transected and peripheral nerve segment was removed. A 2.2-cm biodegradable co-polymer neurotube containing viscous gel (NVR-N-Gel) with survival factors, neuroprotective agents and Schwann cells was placed between the proximal and the distal parts of the transected nerve for reconnection a 2-cm nerve defect. The proximal and distal parts of the nerve were fixed into the neurotube using 10-0 sutures. Ultrasound observation showed growth of the axons into the composite neurotube 2 months after the surgery. Electrophysiological study indicated compound muscle action potentials in nine out of 12 rats, 2-4 months after peripheral nerve reconstructive surgery. The postoperative follow-up (up to 4 months) on the operated rats that underwent peripheral nerve reconstruction using composite co-polymer neurotube, showed beginning of re-establishment of active foot movements. The tube was dissolved and nerve showed complete reconnection. Histological observation of the nerve showed growth of myelinated axons into the site where a 2-cm nerve defect replaced by composite co-polymer neurotube and into the distal part of the nerve. In CONCLUSION: (1) an innovative composite neurotube for reconstruction of significant loss of peripheral nerve segment is described; (2) a viscous gel, containing survival factors, neuroprotective agents and Schwann cells served as a regenerative environment for repair. Further investigations of this reconstructive procedure are being conducted.

Neurol Res. 2004 Mar;26(2):233-9

Phototherapy promotes regeneration and functional recovery of injured peripheral nerve.

Anders JJ, Geuna S, Rochkind S.

Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20854, USA. janders@usuhs.mil

Numerous attempts have been made to enhance and/or accelerate the recovery of injured peripheral nerves. One of the methods studied is the use of phototherapy (low power laser or light irradiation) to enhance recovery of the injured peripheral nerve. A critical analysis of the literature on the employment of phototherapy for the enhancement of the regeneration process of the rat facial and sciatic nerve (after crush injury or transection followed by surgical reconstruction) is provided, together with the description of some of the most suitable basic biological mechanisms through which laser radiation exerts its action on peripheral nerve regeneration.

In Vivo. 2004 Jul-Aug;18(4):489-95.

Effect of Ga-as laser on the regeneration of injured sciatic nerves in the rat.

Bae CS, Lim SC, Kim KY, Song CH, Pak S, Kim SG, Jang CH.

College of Veterinary Medicine, Biotechnology Research Institute, Chonnam National University, Gwangju, Korea.

Laser irradiation is one of the therapeutic methods for the recovery of degenerated peripheral nerves. The aim of the present study was to determine if low-power laser treatment stimulates the regeneration process of damaged nerves. A standardized crush to the sciatic nerve was applied to cause extensive axonal degeneration. After this procedure, low-power infrared laser irradiation was administered transcutaneously to the injured sciatic nerve, 3 minutes daily to each of four treatment groups for 1, 3, 5 and 7 weeks, respectively. A nerve conduction study was done, and a morphological assessment was performed using both light and electron microscopy. With trauma of the nerve, both amplitude of compound motor action potential and nerve conduction velocity decreased significantly compared to the pre-trauma state. Morphologically, the numbers of myelinated axons and degenerated axons were decreased and increased, respectively, compared with the control. Typical aspects were of onion skin-type lamellation, fragmentation, edematous swelling and rarefaction in the myelin sheath. All these parameters recovered almost to the level of the pre-trauma state with laser irradiation, in direct proportion to the time spent for treatment. These results suggest that low-power infrared laser irradiation can relieve the mechanical damage of sciatic nerves and stimulate the regeneration of peripheral nerves.

LASER THERAPY – A NEW MODALITY IN THE TREATMENT OF PERIPHERAL NERVE INJURIES

(Twenty-five years experience from basic science to clinical studies)

S. Rochkind, MD Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel-Aviv University, Tel Aviv, Israel, E-mail: rochkind@zahav.net.il

Since our first publication (Rochkind 1978), we have been studying and testing low power laser irradiation as a means to treat peripheral nerves, using both in vitro and in vivo methods. We have reached the clinical stage and are treating a variety of peripheral nerve injuries. This study is a review of my personal experience over the last twenty-five years in the use of laser therapy in treating these conditions.

I. Influence of Low Power Laser Irradiation on Nerve Cells
A study was done using direct 632.8nm HeNe laser irradiation to determine the effect of focused laser beams on aggregates of rat fetal brain cells and rat adult brain. The direct HeNe laser irradiation 3.6J/cm2 caused a significant amount of sprouting of cellular processes outgrowth in aggregates, compared to small amounts produced by non-irradiated controls. This observation suggests that low power laser irradiation applied to the area of an experimentally injured nerve may induce axonal processes sprouting, thereby improving nerve tissue recovery. The mechanism of low power laser on nerve tissue is not completely understood, but some studies partially explain the photochemical effect of laser irradiation on the biological system. Cytochromes are affected, thereby stimulating redox activity in the cellular respiratory chain, thereby causing increases in ATP production which activates Na+, K+ -ATPase and other ion carriers, thereby increasing cell activation.

II. Animal Studies – influence of laser therapy on the severely injured peripheral nerve
A radiation method for treating lesions in both the peripheral and central nervous systems was proposed in 1978 by Rochkind and modified over the years. The model used in this work was the rat sciatic nerve. Low power laser irradiation then was delivered to the crushed nerve either transcutaneously or directly. The effects of this laser therapy were measured both in the short-term, i.e. minutes and in the long-term, i.e. days and months. Short-term model: direct irradiation of the nerve was done through the open wound directly to the crushed injured nerve and the compound nerve action potential was measured. A variety of wavelengths and powers were applied and 540nm, 632.8nm and 780nm were found most effective (p=0.01). Long-term model: We found electrophysiolgical activity dropped as expected in the non-irradiated nerves following the crush injury, but the use of low power laser irradiation prevented or decreased this phenomenon (p=0.001), both immediately after the crush and in the long term. Furthermore, this investigation showed that when laser treatment was delivered to both the crushed nerve and the corresponding segments of the spinal cord, the recovery time and the quality of regeneration of the crushed sciatic nerve improved, compared to the application of irradiation to the nerve alone. Histological studies supported the electrophysiological findings: low power laser irradiation was found to prevent or decrease scar tissue formation in the injured area. Laser irradiation enhanced axonal sprouting in the crush-injured sciatic nerve, thus accelerating recovery of the severely injured peripheral nerve. In addition, a beneficial effect of low power laser irradiation was found not only in the laser-treated nerve, but in the corresponding segments of the spinal cord as well. Such laser treatment has been found to decrease significantly the degenerative changes in the corresponding neurons of the spinal cord and induce proliferation of neuroglia, both in astrocytes and oligodendrocytes. This suggests a higher metabolism in neurons and a better ability to produce myelin under the influence of laser treatment. Also, low power laser irradiation exerts pronounced systemic effects on severely injured peripheral nerves and corresponding regions of the spinal cord.

III. Double-Blind Randomized Study Evaluating Regeneration of the Rat Sciatic Nerve after Suturing and Post-Operative Laser Therapy
The therapeutic effect of low power laser irradiation on peripheral nerve regeneration after complete transection and direct anastomosis of the rat sciatic nerve was studied recently. A 780nm laser wavelength was applied transcutaneously 30 minutes daily for 21 consecutive days to corresponding segments of the spinal cord and to the injured sciatic nerve immediately after closing the wound. Positive somato-sensory evoked responses were found in 55% of the irradiated rats and in 11% of the non-irradiated rats. Immuno-histochemical staining in the laser-treated group showed more intensive axonal growth and better quality of the regenerative process due to an increased number of large and medium diameter axons. IV. Clinical Pilot Studies The group of patients who were treated in the Department of Neurosurgery at Tel Aviv Sourasky Medical Center had been suffering from severe peripheral nerve and brachial plexus injuries for more than two years. Each of the 59 patients received laser treatment CW, 780nm, five hours daily for 21 consecutive days with the use of a laser system specially developed for our treatment method. Criterion for laser treatment in these cases was as follows: patients who suffered from partial motor and sensory disturbances and where surgery was not indicated. Fifty-six percent of the laser-treated patients showed good to excellent results in their motor function. V. Clinical Double-Blind Placebo-Controlled, Randomized Study of Low Power Laser in the Treatment of Peripheral Nerve Injures Since our previous pilot clinical results were positive, a final evaluation of the response to treatment was in order. Therefore, we performed a double-blind, placebo-controlled randomized study of patients who had been suffering from incomplete peripheral nerve and brachial plexus injuries from 6 months up to several years after injury. The protocol of this study was done with the permission of the Helsinki Committee of the Tel Aviv Sourasky Medical Center and with the approval of the Ministry of Health of Israel and by a grant from the Rehabilitation Department of the Ministry of Defence of Israel. The study evaluated the functional recovery of these patients after undergoing low power laser or placebo treatment. Recovery was classified by comparing each of the deficits present before and after surgery. The post-laser or post-placebo grade was determined by the change in strength compared to the pretreatment levels. In almost all cases, the level of motor function was minimal to poor pre-treatment. In the laser-treated group, statistically significant improvement was found in motor functional activity P=0.0001, compared to the placebo group). The electrophysiological findings also showed statistically significant improvement in the laser-treated group. Our twenty-five years of experience indicates that Laser Therapy is a low-cost, non-invasive method and will be recognized as standard additional treatment for improving the functional recovery of patients with peripheral nerve and brachial plexus injuries. According to our clinical experience, the main advantages of Laser Therapy are the enhancement and acceleration of the recovery of injured nerve tissue. The therapeutic results show that an objective progressive improvement appears in nerve function, leading to a significant and earlier recovery.

J Reconstr Microsurg. 2001 Feb;17(2):133-7; discussion 138.

Double-blind randomized study evaluating regeneration of the rat transected sciatic nerve after suturing and postoperative low-power laser treatment.

Shamir MH, Rochkind S, Sandbank J, Alon M.

Koret School of Veterinary Medicine, Hebrew University of Jerusalem.

This double-blind randomized study evaluated the therapeutic effect of low-power laser irradiation (LPLI) on peripheral nerve regeneration, after complete transection and direct anastomosis of the rat sciatic nerve. After this procedure, 13 of 24 rats received postoperative LPLI, with a wavelength of 780 nm laser, applied transcutaneously, 30 min daily for 21 consecutive days, to corresponding segments of the spinal cord and to the injured sciatic nerve. Positive somatosensory evoked responses were found in 69.2 percent of the irradiated rats (p = 0.019), compared to 18.2 percent of the non-irradiated rats. Immunohistochemical staining in the laser-treated group showed an increased total number of axons (p = 0.026), and better quality of the regeneration process, due to an increased number of large-diameter axons (p = 0.021), compared to the non-irradiated control group. The study suggests that postoperative LPLI enhances the regenerative processes of peripheral nerves after complete transection and anastomosis.

Lasers Med Sci. 2003;18(2):83-8

No effect of GA-AS (904 nm) laser irradiation on the injured rat sciatic nerve.

Bagis S, Comelekoglu U, Coskun B, Milcan A, Buyukakilli B, Sahin G, Ozisik S, Erdogan C.

Mersin University, Adana, Turkey. seldabagis@hotmail.com

We evaluated the electrophysiological and histopathological effects of low-energy gallium arsenide (904 nm) laser irradiation on the intact skin injured rat sciatic nerve. Twenty-four male Wistar rats were divided into three groups ( n=8 each). At the level of proximal third of the femur the sciatic nerve was crushed bilaterally with an aneurysm clip (Aesculap FE 751, Tuttingen, Germany) for half a second. A gallium arsenide laser (wavelength 904 nm, pulse duration 220 ns, peak power per pulse 27 W, spot size 0.28 cm2, pulse repetition rate 16, 128 and 1000 Hz; total applied energy density 0.31, 2.48 and 19 J/cm2) was applied to the right sciatic nerve for 15 min daily at the same time on 7 consecutive days. The same procedure was performed on the left sciatic nerve of same animal, but without radiation emission, and this was accepted as control. Compound muscle action potentials were recorded from right and left sides in all three groups before surgery, just at the end of injury, at the 24th hour and on the 14th and 21st days of injury in all rats using a BIOPAC MP 100 Acquisition System Version 3.5.7 (Santa Barbara, USA). BIOPAC Acknowledge Analysis Software (ACK 100 W) was used to measure CMAP amplitude, area, proximal and distal latency, total duration and conduction velocity. Twenty-one days after injury, the rats were sacrificed. The sciatic nerves of the operated parts were harvested from the right and left sides. Histopathological evaluation was performed by light microscopy. Statistical evaluation was done using analysis of variance for two factors (right and left sides) repeated-measures (CMAP variables within groups) and the Tukey-Kramer Honestly Significant Difference test (CMAP variables between laser groups). The significance was set at p < 0.05. No statistically significant difference (p > 0.05) was found regarding the amplitude, area, duration and conduction velocity of CMAP for each applied dose (0.31, 2.48 and 19 J/cm2) on the irradiated (right) side and the control (left) side, or between irradiated groups. Twenty-one days after injury there were no qualitative differences in the morphological pattern of the regenerated nerve fibres in either irradiated (0.31, 2.48 and 19 J/cm2) or control nerves when evaluated by light microscopy. This study showed that low-energy GaAs irradiation did not have any effect on the injured rat sciatic nerve.

J. Käs . PNAS. 2002; 99: 16024-16028

Guiding neuronal growth with light

A. Ehrlicher, T. Betz, B. Stuhrmann, D. Koch, V. Milner, M. G. Raizen,

We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, we place a laser spot in front of a chosen area of the nerve’s leading edge, promoting growth into the beam focus. This allows us to guide neuronal turns as well as enhance growth. The power of our laser has been selected so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers, which uses large optical forces to manipulate entire structures. Our results therefore open a new avenue to controlling neuronal growth in vitro and in vivo with a simple, non-contact technique. Currently we have been using 800nm with continuous application of powers ranging from 20 to 130 mW over a circular area of 1 to 4 um in radius. Recently we’ve developed and active feedback mechanism to trace the contour of the growth cone and subsequently raster the beam image upon that, instead of the pure beam profile we had used previously.
(Abstract supplied by Allen Ehrlicher, main author)

Neurosci Lett. 2003 Jun 26;344(2):71-4.

 

Growth-associated protein-43 is elevated in injured rat sciatic nerve after low power laser irradiation.

Shin DH, Lee E, Hyun JK, Lee SJ, Chang YP, Kim JW, Choi YS, Kwon BS.

Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea.

Low power laser irradiation (LPLI) has been used in the treatment of peripheral nerve injury. In this study, we verified its therapeutic effect on neuronal regeneration by finding elevated immunoreactivities (IRs) of growth-associated protein-43 (GAP-43), which is up-regulated during neuronal regeneration. Twenty Sprague-Dawley rats received a standardized crush injury of the sciatic nerve, mimicking the clinical situations accompanying partial axonotmesis. The injured nerve received calculated LPLI therapy immediately after injury and for 4 consecutive days thereafter. The walking movements of the animals were scored using the sciatic functional index (SFI). In the laser treated rats, the SFI level was higher in the laser treated animals at 3-4 weeks while the SFIs of the laser treated and untreated rats reached normal levels at 5 weeks after surgery. In immunocytochemical study, although GAP-43 IRs increased both in the untreated control and the LPLI treated groups after injury, the number of GAP-43 IR nerve fibers was much more increased in the LPLI group than those in the control group. The elevated numbers of GAP-43 IR nerve fibers reached a peak 3 weeks after injury, and then declined in both the untreated control and the LPLI groups at 5 weeks, with no differences in the numbers of GAP-43 IR nerve fibers of the two groups at this stage. This immunocytochemical study using GAP-43 antibody study shows for the first time that LPLI has an effect on the early stages of the nerve recovery process following sciatic nerve injury.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002 Jan;93(1):27-34.

 

Low-level laser effect on neural regeneration in Gore-Tex tubes.

Miloro M, Halkias LE, Mallery S, Travers S, Rashid RG.

Department of Surgery, Division of Oral and Maxillofacial Surgery, University of Nebraska Medical Center, Omaha 68198-5180, USA.

PURPOSE: The purpose of this investigation was to determine the effects of low-level laser (LLL) irradiation on neural regeneration in surgically created defects in the rabbit inferior alveolar nerve.

STUDY DESIGN: Five adult female New Zealand White rabbits underwent bilateral exposure of the inferior alveolar nerve. A 6-mm segment of nerve was resected, and the nerve gap was repaired via entubulation by using a Gore-Tex conduit. The experimental side received 10 postoperative LLL treatments with a 70-mW gallium-aluminum-arsenide diode at 4 sites per treatment. At 15 weeks after surgery, the nerve segments were harvested bilaterally and prepared for light microscopy. Basic fuchsin and toluidine blue were used to highlight myelinated axons. The segments were examined histomorphometrically by using computer analysis to determine mean axonal diameter, total fascicular surface area, and axonal density along the repair sites.

RESULTS: Gross examination of all nerves showed intact neural bundles with variable degrees of osseous remodeling. Light microscopic evaluation revealed organized regenerated neural tissue in both groups with more intrafascicular perineural tissue in the control group. Histomorphometric evaluation revealed increased axonal density in the laser treated group as compared with the control.

CONCLUSIONS: LLL irradiation may be a useful noninvasive adjunct to promote neuronal wound healing in surgically created defects repaired with expanded polytetrafluoroethylene entubulation.

Spine (Phila Pa 1976). 1990 Jan;15(1):6-10.

Spinal cord response to laser treatment of injured peripheral nerve.

Rochkind S, Vogler I, Barr-Nea L.

Department of Neurosurgery, Ichilov Hospital, Tel-Aviv Medical Center, Israel.

Abstract

The authors describe the changes occurring in the spinal cord of rats subjected to crush injury of the sciatic nerve followed by low-power laser irradiation of the injured nerve. Such laser treatment of the crushed peripheral nerve has been found to mitigate the degenerative changes in the corresponding neurons of the spinal cord and induce proliferation of neuroglia both in astrocytes and oligodendrocytes. This suggests a higher metabolism in neurons and a better ability for myelin production under the influence of laser treatment.

Lasers Surg Med. 1989;9(2):174-82.

Systemic effects of low-power laser irradiation on the peripheral and central nervous system, cutaneous wounds, and burns.

Rochkind S, Rousso M, Nissan M, Villarreal M, Barr-Nea L, Rees DG.

Department of Neurosurgery, Tel Aviv Medical Center, Ichilov Hospital, Israel.

Abstract

In this paper, we direct attention to the systemic effect of low-power helium-neon (HeNe) laser irradiation on the recovery of the injured peripheral and central nervous system, as well as healing of cutaneous wounds and burns. Laser irradiation on only the right side in bilaterally inflicted cutaneous wounds enhanced recovery in both sides compared to the nonirradiated control group (P less than .01). Similar results were obtained in bilateral burns: irradiating one of the burned sites also caused accelerated healing in the nonirradiated site (P less than .01). However, in the nonirradiated control group, all rats suffered advanced necrosis of the feet and bilateral gangrene. Low-power HeNe laser irradiation applied to a crushed injured sciatic nerve in the right leg in a bilaterally inflicted crush injury, significantly increased the compound action potential in the left nonirradiated leg as well. The statistical analysis shows a highly significant difference between the laser-treated group and the control nonirradiated group (P less than .001). Finally, the systemic effect was found in the spinal cord segments corresponding to the crushed sciatic nerves. The bilateral retrograde degeneration of the motor neurons of the spinal cord expected after the bilateral crush injury of the peripheral nerves was greatly reduced in the laser treated group. The systemic effects reported here are relevant in terms of the clinical application of low-power laser irradiation as well as for basic research into the possible mechanisms involved.

Health Phys. 1989 May;56(5):687-90.

New biological phenomena associated with laser radiation.

Belkin M, Schwartz M.

Goldschleger Eye Research Institute, Tel-Aviv University, Sackler School of Medicine, Tel-Hashomer, Israel.

Abstract

Low-energy laser irradiation produces significant bioeffects. These effects are manifested in biochemical, physiological and proliferative phenomena in various enzymes, cells, tissues, organs and organisms. Examples are given of the effect of He-Ne laser irradiation in preventing the post-traumatic degeneration of peripheral nerves and the postponement of degeneration of the central nervous system. The damage produced by similar radiant exposures to the corneal epithelium and endothelium is also described. It is suggested that the mechanism of laser/tissue interaction at these low levels of radiant exposure is photochemical in nature, explaining most of the characteristics of these effects. These low-energy laser bioeffects are of importance on a basic scientific level, from a laser safety aspect and as a medical therapeutic modality.

Lasers Surg Med. 1987;7(5):441-3.

Response of peripheral nerve to He-Ne laser: experimental studies.

Rochkind S, Nissan M, Barr-Nea L, Razon N, Schwartz M, Bartal A.

Neurosurgery Department, Tel-Aviv Medical Center, Ichilov Hospital, Israel.

Abstract

Low-energy He-Ne laser irradiation (LELI) was found to affect the electric activity and morphology in both intact and severely injured peripheral nerves in rats. Action potential (AP) in the healthy nerve increased by 33% following a single transcutaneous irradiation. Similar irradiation in crushed nerves caused AP to increase significantly over the AP of nonirradiated crushed nerve. Morphological observations revealed that a laser-irradiated injured nerve had diminished scar tissue as compared to an injured but not an irradiated nerve.