Photomedicine and Laser Surgery

Effects of Power Densities, Continuous and Pulse Frequencies, and Number of Sessions of Low-Level Laser Therapy on Intact Rat Brain

Aug 2006, Vol. 24, No. 4: 458-466

Sanja Ilic , M.D. Photothera Inc., Carlsbad, California.

Sandra Leichliter , M.Sc. Photothera Inc., Carlsbad, California.

Jackson Streeter , M.D. Photothera Inc., Carlsbad, California.

Amir Oron , M.D. Photothera Inc., Carlsbad, California.

Luis DeTaboada , M.S.E.E. Photothera Inc., Carlsbad, California.

Dr. Uri Oron , Ph.D. Photothera Inc., Carlsbad, California.

Objective: The aim of the present study was to investigate the possible short- and long-term adverse neurological effects of low-level laser therapy (LLLT) given at different power densities, frequencies, and modalities on the intact rat brain. Background Data: LLLT has been shown to modulate biological processes depending on power density, wavelength, and frequency. To date, few well-controlled safety studies on LLLT are available.

Methods: One hundred and eighteen rats were used in the study. Diode laser (808 nm, wavelength) was used to deliver power densities of 7.5, 75, and 750 mW/cm² transcranially to the brain cortex of mature rats, in either continuous wave (CW) or pulse (Pu) modes. Multiple doses of 7.5 mW/cm² were also applied. Standard neurological examination of the rats was performed during the follow-up periods after laser irradiation. Histology was performed at light and electron microscopy levels.

Results: Both the scores from standard neurological tests and the histopathological examination indicated that there was no long-term difference between lasertreated and control groups up to 70 days post-treatment. The only rats showing an adverse neurological effect were those in the 750 mW/cm² (about 100-fold optimal dose), CW mode group. In Pu mode, there was much less heating, and no tissue damage was noted.

Conclusion: Long-term safety tests lasting 30 and 70 days at optimal 10× and 100× doses, as well as at multiple doses at the same power densities, indicate that the tested laser energy doses are safe under this treatment regime. Neurological deficits and histopathological damage to 750 mW/cm² CW laser irradiation are attributed to thermal damage and not due to tissue–photon interactions.

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 shortterm,i.e. minutes and in the long-term, i.e. days and months. Short-term model: direct irradiationof the nerve was done through the open wound directly to the crushed injured nerve and thecompound nerve action potential was measured. A variety of wavelengths and powers wereapplied 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: lowpower 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 lasertreated 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. Fiftysix 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 doubleblind,
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 motorfunction was minimal to poor pretreatment.
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.

An Innovative Approach To Induce Regeneration And The Repair Of Spinal Cord Injury
Laser Therapy.1997; 9 (4): 151.
Rochkind S, Shahar A. Nevo Z.
An Israeli research group has investigated an innovative method of repairing injured spinal cords.
In a rat model the spinal cords were transected in 31 animals (between T7/T8). In vitro
constructed composite implants were used in the transected area. These implants contained
embryonal spinal cord neuronal cells dissociated from rat fetuses, cultured on biodegradable
microcarriers. After being embedded in hyaluronic acid the implants were ready to be placed into
the injured area. The whole lesion area was covered with a thin coagulated fibrin-based
membrane. Control animals underwent the same laminectomy but did not receive any implant. In
all animals the wound was closed normally. Laser therapy was started immediately after surgery.
It was continued daily for two weeks using 780 nm, 200 mW, 30 minutes daily. One group
received the implant but no laser. During the 3-6 months follow up, 14 of the 15 animals that
received laser (A) showed different degrees of active movements in one or both legs, compared
to 4 of 9 animals in the group who had received implants but no laser (B). In the group receiving
no implant and no laser (C), 1 out of 7 showed some motor movements in one leg.
Somatosensory evoked potentials were elicited in 10 of the 15 rats in group A at three months,
and on one side in one animal in group B. Axon sprouting was observed as soon as three days
post surgery, in group A only.

New Hope For Patients With Spinal Cord Injuries
An Innovative Approach To Induce Regeneration And The Repair Of Spinal Cord Injury
Rochkind S, Shahar A. Nevo Z.
Laser Therapy.1997; 9 (4): 151.
An Israeli research group has investigated an innovative method of repairing injured spinal cords.
In a rat model the spinal cords were transected in 31 animals (between T7/T8). In vitro
constructed composite implants were used in the transected area. These implants contained
embryonal spinal cord neuronal cells dissociated from rat fetuses, cultured on biodegradable
microcarriers. After being embedded in hyaluronic acid the implants were ready to be placed into
the injured area. The whole lesion area was covered with a thin coagulated fibrin-based
membrane. Control animals underwent the same laminectomy but did not receive any implant. In
all animals the wound was closednormally. Laser therapy was started immediately after surgery.
It was continued daily for two weeks using 780 nm, 200 mW, 30 minutes daily. One group
received the implant but no laser. During the 3-6 months follow up, 14 of the 15 animals that
received laser (A) showed different degrees of active movements in one or both legs, compared
to 4 of 9 animals in the group who had received implants but no laser (B). In the group receiving
no implant and no laser (C), 1 out of 7 showed some motor movements in one leg.
Somatosensory evoked potentials were elicited in 10 of the 15 rats in group A at three months,
and on one side in one animal in group B. Axon sprouting was observed as soon as three days
post surgery, in group A only.

Guiding Neuronal Growth With Light
A. Ehrlicher, T. Betz, B. Stuhrmann, D. Koch, V. Milner, M. G. Raizen,
J. Käs . PNAS. 2002; 99: 16024-16028
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 actinpolymerization-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)

Transplantation Of Embryonal Spinal Cord Nerve Cells Cultured On Biodegradable
Microcarriers Followed By Low Power Laser Irradiation For The Treatment Of Traumatic Paraplegia In Rats
Neurol Res. 2002 Jun;24(4):355-60.
Rochkind S, Shahar A, Amon M, Nevo Z.
Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Israel.
rochkind@zahav.net.il
This pilot study examined the effects of composite implants of cultured embryonal nerve cells and laser irradiation on the regeneration and repair of the completely transected spinal cord.
Embryonal spinal cord nerve cells dissociated from rat fetuses and cultured on biodegradable
microcarriers and embedded in hyaluronic acid were implanted in the completely transected
spinal cords of 24 adult rats. For 14 consecutive post-operativedays, 15 rats underwent low
power laser irradiation (780 nm, 250 mW), 30 min daily.
Eleven of the 15 (73%) showed different degrees of active leg movements and gait performance,
compared to 4 (44%) of the 9 rats with implantation alone. In a controlgroup of seven rats with
spinal cord transection and no transplantation or laser, six (86%) remained completely paralyzed.
Three months after transection, implantation and laser irradiation, SSEPs were elicited in 69% of
rats (p = 0.0237) compared to 37.5% in the nonirradiated group. The control group had no SSEPs response. Intensive axonal sprouting occurred in the group with implantation and laser. In the control group, the transected area contained proliferating fibroblasts and blood capillaries only. This suggests:

1. These in vitro composite implants are a regenerative and reparative source for
reconstructing the transected spinal cord.

2. Post-operative low power laser irradiation enhances
axonal sprouting and spinal cord repair.

Growth-Associated Protein-43 Is Elevated In The 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.
Neurosci Lett. 2003 Jun 26;344(2):71-4.
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.

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

Anders J.J., et al.
Lasers in Surgery and Medicine 13:72-82 (1993), ©Wiley-Liss, Inc.
"Low Power Laser Irradiation Alters the Rate of Regeneration of the Rat Facial
Nerve"
Low power laser irradiation has been reported to cause biological effects due to the
photochemical and/or photophysical action of the radiation. This study determined quantitatively if transcutaneous low power laser irradiation can affect the regeneration of the rat facial nerve. The facial nerve was crushed unilaterally in anesthetized rats and transcutaneously irradiated daily with a laser beam directed at the area of the crush injury. Laser treatment began on the day of the crush injury and was continued daily for 7,8, or 9 days. Preliminary experiments determined the most effective wavelength, laser power, length of irradiation, and treatment schedule.

The wavelengths examined were 361, 457, 514, 633, 720, and 1064. The laser powers and lengths of irradiation examined ranged from 8.5 to 40 mW and 13 to 120 min. Irradiation treatment was done daily, on alternating days and on the first 4 days postcrush. The most effective laser parameters for the low power treatment included daily irradiation with a helium-neon (HeNe) or argon pumped tunable dye laser a wavelength of 633 nm, with a power of 8.5 mW for 90 minutes (45.9 J, 162.4 J/cm2). The number of horseradish peroxide (HRP) labeled neurons in the facial motor nucleus was used as an assay of the degree of regeneration. In rats in which the facial nerve was crushed but not irradiated, the average number of HRP labeled neurons in the facial nucleus was 22 on day 7 postcrush, 54 on day 8, 116 on day 9, and 1149 on day 10. After HeNe or argon pumped tunable dye laser irradiation, the average number of HRP-labeled neurons increased to 34 on day 7 postcrush, 148 on day 8, and 1725 on day 9. There was a statistically significant difference between the control and irradiated rats on day 9 postcrush (p<0.01). These data indicate that transcutaneous low power irradiation with the lasers and parameters involved in this study increased the rate of regeneration of rat facial nerve following crush injury.

Bernal G., et al.
Laser Therapy Vol.5, No.2, 79-87, 1993 © John Wiley & Sons, Ltd.
"Helium neon and diode laser therapy is an effective adjunctive therapy for facial
paralysis"
This study presents our six-year experience in laser therapy for rehabilitation of facial paralysis.
Mixed laser irradiation using a 904 nm diode GaAs and 632.8 was utilized. Laser irradiation was performed on the range of facial nerve ramifications in eight different places, 5 min on each place, four times a week. No other medicine was used if the patient arrived 48 h after having the lesion.
When the patient arrived after the first week, meticoren was utilized as a supplement, a dosage of 40 mg per day, for seven days. Based on our experience, the patients who are more inclined to attend treatment sessions are those who have been suffering from paralysis for more that a month and who have submitted to other kinds of treatments with negative results. They were even offered surgery. For these patients, we have required up to maximum of 30 sessions and have achieved 100% recovery, even with patients who have had the lesion for three or six months. Patients who attended therapy within two weeks after suffering the paralysis recovered 100% with no additional medication -only laser therapy. With these patients we needed a maximum of 15 sessions. LLLT is presented as a safe, noninvasive, easy to apply and comparatively side-effect-free modality offering a complementary and effective tool in the treatment of facial paralysis.

Murakami F. et al.
Laser Therapy 5; 131-135, 1993 © John Wiley & Sons, Ltd.
"Diode low reactive level laser therapy and stellate ganglion block compared in the treatment of facial palsy"
In 52 patients who presented with peripheral facial paralysis, 26 received stellate ganglion block therapy, 11 received infrared diode laser low reactive level laser therapy, and 15 received a combination of both of the above. The data were analyzed to compare the effectiveness of the three regiments. Those patients who received only LLLT or the combination of LLLT with SGB showed a similar overall recovery from the paralysis compared to those treated with SGB alone.
The group who received LLLT only also demonstrated a slightly better initial improvement in paralysis scores. No serious side effects were reported in the LLLT group. Taking the above data into consideration, the authors recommend diode laser therapy as a suitable single or adjunctive therapy for facial paralysis which is relatively easy and painless to apply, requires less technical skill, compared with SGB, and has no side-effects.

Midamba E.D., et al.
Laser Therapy 5; 125-129, 1993 © John Wiley & Sons, Ltd.
"Low reactive-level 830 nm GaAlAs diode laser therapy successfully accelerates regeneration of peripheral nerves in human"
Forty patients with short and long-term neurosensory impairment following perioral nerve injuries are presented in this study. Assessment of their sensory level was undertaken using a variety of nerve tests, one of them was a visual analog scale for registration of sensitivity level prior to and after 10 treatment sessions and additionally for 21 of the 40 patients after 20 treatment sessions.
Low level laser therapy was applied using GaAlAs 830 nm, 70 mW continuous wave. Dose of 6.0 J/cm2 was standardized for all patients. Improvement of the eight patients with clinical symptoms of less than 1 year after 10 treatments, was between 40-90% and after 20 treatments between 60-80% for the three patients who continued with the treatment. In 32 of the 40 patients with clinical symptoms of more than 1 year in duration, their improvement was estimated at between 40 and 80%, 21 patients completed 20 treatment sessions and the end results were between 60-90%. This was an uncontrolled clinical study of LLLT on perioral nerve injuries and demonstrated the effectiveness of GaAlAs laser on the nerve involved when applied to the nerve trunk and terminal endings. Although controlled research into actual mechanisms and pathways is needed, the preliminary findings are very promising.

Snyder S.K., et al.
Lasers in Surgery and Medicine 31:216-222, 2002 © Wiley-Liss, Inc.
"Quantitation of Calcitonin Gene-Related Peptide mRNA and Neuronal Cell Death in Facial Motor Nuclei Following Axotomy and 633 nm Low Power Laser Treatment"
A persistent increase in calcitonin gene-related peptide immunoreactivity in motor neurons may serve as an indicator for regeneration after peripheral nerve injury. We examined the effects of low power laser treatment on axotomy-induced changes in alpha-CGRP mRNA and long-term neuronal survival in facial motor neurons. A quantitative reverse transcriptase-polymerase chain reaction assay for alpha-CGRP mRNA was used to detect changes in the response to axotomy and laser irradiation. Cell counts of neurons in injured and non-injured facial motor nuclei of lasertreated and non-treated rats were done to estimate neuronal survival. A 10-fold increase in mRNA for alpha-CGRP at 11 days post-transsection and an almost threefold increase in neuronal survival at 6-9 months post-transsection were found in 633nm light treated rats. These findings demonstrate that 633nm laser light upregulates CGRP mRNA and support theory that laser irradiation increases the rate of regeneration, target reinnervation, and neuronal survival of the axotomized neuron.

Brugnera A. Jr., et al.
Lasers in Dentistry VI, SPIE Vol. 3910, 2000
"Low-reactive level laser treatment in facial paralysis"
This study was carried out with a 41-year-old female patient with facial paralysis as a
consequence of facial nerve injury during neurosurgery. Low-reactive level laser treatment with a diode laser of 830nm, 40 mW, continuous wave, spot area 3 mm2, was applied twice a week for 2 weeks, then 1 weekly session