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Original Article
The Efficacy of Low-Level Laser Therapy and Low Intensity Pulsed
Ultrasound on Functional Recovery among Experimentally Induced
Peripheral Nerve Injury in Wistar Rats
Dhivya S1, Ramana K2, Prathap Suganthira Babu3, Lavanya Prathap4, Mydhili
Govindarasu5, Kumaresan A6, Vignesh Srinivasan7
1Post graduate, 2,7Assistant Professor, 3,6Professor, Saveetha College of Physiotherapy, SIMATS,
4Associate Professor, Department of Anatomy, 5Assistant Professor, BRULAC,
Saveetha Dental College and Hospital, Chennai.
How to cite this article: Dhivya S, Ramana K, Prathap Suganthira Babu et. al. The Efficacy of Low-Level Laser
Therapy and Low Intensity Pulsed Ultrasound on Functional Recovery among Experimentally Induced Peripheral
Nerve Injury in Wistar Rats. Indian Journal of Physiotherapy and Occupational Therapy / Volume 18 Special
Issue 2024;
Abstract
Background: A variety of etiological factors that lead to peripheral nerve injury include crushing, traction, long
fractures in bones, cutting injuries, entrapment neuropathies, infection, inflammation, and tumours of varying
degrees. LIPUS has a variety of biological effects on tissues, including speeding up the regeneration of soft tissues,
and lowering inflammatory reactions. In LLLT, low-level laser light with a wavelength of 808 nm is employed to
trigger a biological reaction.
Purpose: The objective of this study was to evaluate the functional recovery of low-level laser therapy (LLLT) and
low-intensity pulsed ultrasound (LIPUS) treating the sciatic nerve in rats.
Methods: Using simple random sampling method and the inclusion and exclusion criteria, 18 rats in total were
chosen. A mechanical crush will be performed for 30 seconds using haemostatic forceps. The study’s rats were
divided into 3 groups at random: Group A received ultrasound for 21 days; Group B received laser for 21 days;
and Group C received no treatment. The toe spread assay was used to assess functional recovery 3 weeks after
surgery. Result: All three groups are statistically significant (p<0.005) when analysed using one-way ANOVA
during intervention period (7th, 24th, 21st day) but LLLT & UST has shown better improvement in their functional
index than the control group.
Conclusion: According to the study’s findings, LLLT significantly outperforms LIPUS in improving the functional
recovery of an experimentally induced rat model.
Keywords: animal study, physiotherapy intervention, nerve regeneration, functional recovery and sciatic nerve
injury.
Introduction
Common peripheral nerve injuries frequently
prevent peripheral nerve axons from regenerating
considerably, and only around 10% of patients are
able to fully recover their function.1 The reason for
the poor functional results is typically attributed to
Corresponding Author: Prathap Suganthirababu, Professor, Saveetha College of Physiotherapy, SIMATS, Chennai.
E-mail: emailprathap@gmail.com
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the sluggish axon regeneration process.2 The severity
of this problem was brought home by a research
done in the US, which found that 50,000 or so people
experience severe peripheral nerve injuries annually,
making it a major public health issue.3 Axonotmesis,
neurotmesis, and neuropraxia were the three primary
forms of nerve injuries discovered by a recent analysis
in Brazil that looked at 456 cases of nerve damage.4
Wallerian degeneration manifests in the distal stump
when nerve fibre continuity is broken.
Since nerve innervation is essential to the structure
and appropriate operation of muscles, peripheral
nerve injury has a significant impact on skeletal
muscle.5 Therefore, enhancing axon regeneration
is a viable strategy for treating peripheral nerve
injury. After such injuries, a number of rehabilitation
strategies have been proposed to aid functional
recovery. For instance, a herniated disc might directly
compress and irritate the spinal canal’s nerve roots.
Pain, numbness, tingling, and muscle weakness can
all be symptoms of nerve compression and irritation.
Despite the fact that recovery is mostly effective, it
frequently occurs slowly and insufficiently, which
leaves the patient in despair and results in large
expenses for the community as a whole.6
A damaged peripheral nerve can lead to
functional limitations or a lack of sensation in the
afflicted location. Glial cells, also known as Schwann
cells, play a crucial role in the regeneration of
peripheral nerves. They are a part of the peripheral
nervous system and are in charge of assisting and
directing nerve regeneration. It might discuss
topics like how neurotrophic factors are secreted by
Schwann cells, how they affect nerve cell survival and
proliferation, and how they affect the entire process
of nerve healing.
Wistar rats were used in this investigation
because they are simple to handle, are reasonably
priced, and have human-like anatomy, physiology,
and peripheral nerve regeneration. This therapeutic
technique, called low-level laser therapy (LLLT),
has mostly been utilized to promote regeneration
and speed up the functional recovery of peripheral
nerves. Crush, compression, stretching, avulsion,
and division are the most frequent causes of
peripheral nerve harm. Despite the use of advanced
and contemporary techniques for treatment
and reconstruction, morphologic and functional
regeneration is rarely fully achieved because of the
influence of variables like the type and degree of
damage, the length of denervation, the type and
diameter of the damaged nerve fibres, age, and other
specific variables.
Even while some degree of healing is possible, it
frequently happens slowly and insufficiently, leaving
the patient feeling hopeless and placing a heavy
financial burden on the community as a whole.7 The
severity or extent of a peripheral nerve damage has
a significant impact on the recovery process.8 Even
with microsurgical restoration, functional recovery
following peripheral nerve damage is typically
inadequate, especially in cases when significant nerve
trunks are injured. Low-Level Laser Therapy (LLLT)
induces a photochemical reaction in cells known as
“biostimulation” or “photobiomodulation.”
Peripheral neuropathies have so far not been
successfully treated with medication, including
several neurotrophic drugs.9 Even with improvements
in surgical and medical procedures, peripheral nerve
injury frequently does not completely recover. The
poor reinnervation of motor and sensory target areas
is one of the major obstacles to obtaining maximum
recovery.10 In order to address this; novel treatment
approaches that can quicken and improve the healing
process can show therapeutic promise. In this study,
the effectiveness of laser therapy and ultrasound
in hastening functional recovery in rats with right
sciatic nerve injury is being evaluated.
Aim
The aim of this study is to evaluate and compare
the efficacy of low-level laser therapy and low-
intensity pulsed ultrasound in promoting functional
recovery in rats with peripheral nerve injury.
Materials and Methods
This study was conducted from July 2022 to
November 2022. It was an experimental study
conducted on 18 male adult Wistar rats with
experimentally induced peripheral nerve injury, aged
3 months and weighing 150-300g, was chosen for the
study. They were kept in cages with one other animal
apiece, fed on pellets, and given water. After being
weighed, the rats were divided into three groups of
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six at random. Every animal underwent the exact
identical surgical treatment. Groups 1 and 2 received
ultrasound and laser treatment, whereas Group 3
received a control group.
Inclusion criteria
Wistar rats between the ages of 6 months (0.5)
and 48 months (4.0) were included.
Only male wistar rats were included.
Rats that are physically active were included.
Exclusion Criteria
Rats with physical abnormalities and skin issues
are excluded.
Rats that exhibit psychological distress or
aggression are prohibited.
Outcome measure
Assessment was performed at baseline
preoperative, 7th, 14th, and 21st postoperative days.
Toe Spread Assay was used as an outcome measure.
Procedure
All 18 animals were given intramuscular
and intraperitoneal injections of 5% ketamine
hydrochloride (70 mg/kg body weight) and 2%
xylazine hydrochloride (10 mg/kg body weight)
in a 1:4 ratio to induce anaesthesia. Meloxicam was
administered subcutaneously in a dose of 1 mg/kg
to treat the pain. By carefully dissecting between the
gluteus maximus and quadriceps muscles, a 3 cm
long postero-lateral longitudinal thigh incision was
made, exposing the right sciatic nerve. The skin hair
was shaved to make the sciatic nerve area visible, and
54 N of crush injury was then caused for 30 seconds
using hemostatic forceps.
Ultrasound group (Group 1)
In this investigation, the crush site was the only
target for the pulsed-wave ultrasound, which covered
a range of frequencies, durations, and spatial peaks
with time-averaged intensities. With a finger probe
and a pulsed intensity of 0.4 W/cm2 in a 1:1 ratio, the
ultrasound used had a frequency of 1 MHz and was
applied transcutaneously. The treatment area over
the crush injury site was treated for 4 minutes. The
transducer was placed around 2 to 3 cm distant from
the sutured area over the glove water bed in order to
enable effective transmission of ultrasound into the
animal’s lower extremities. The treatment focused on
the site of the crush injury and started on the second
post - operative day (postoperative day 2). It lasted
for 21 days in total. On days 7, 14, and 21 following
surgery, the toe spread assay was carried out and
compared to the pre-injury assessment to gauge
functional recovery.
Laser group (Group 2)
For low-level laser irradiation in this study,
a portable Aluminium Gallium Arsenide Laser
Diode was employed. The laser met the following
requirements: an energy density of 3 J/cm2, an area
length, width, and tissue depth of 1 cm, a wavelength
of 808 nm (infrared light), a power of 200 mW, and
continuous mode. Each session’s exposure period
was set to 60 seconds. Patients in the experimental
group 2 had laser irradiation with a focus on the nerve
damage location that had been surgically identified.
The contact point approach was used to apply a laser
pen at a 90-degree angle to the skin during the first
21 days following surgery. According to Monte-Raso,
this method proved quantitative, trustworthy, and
reproducible in rat sciatic nerve operating settings.
Control group (Group 3)
Control group also underwent the same surgical
procedure and no treatment was provided to the
animals in the control group.
Assessment of nerve functional recovery
Toe spread assay
On the seventh, fourteenth, and twenty-first days
after the right sciatic nerve lesion, the toe spread
measurement was performed to gauge the degree
of functional recovery. Both the experimental and
control animals had water-soluble black ink painted
on their hind limbs, and they were both free to travel
on a white paper track, leaving their prints in their
behind. At first, the measurement, application of the
ultrasound, and toe spread assay were all completed
on the same day. Since the study was coded by
animal number without mentioning the groups they
belonged to, one of the authors measured the walking
track using an objective manner to ensure impartial
evaluation. The distance between the first and fifth
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toes was used to determine the toe spread index
for each experimental group, and the results were
compared to those of the laser and control groups.
ANOVA analyses were performed on the toe spread
assay with a 5% (p0.05) between-groups significant
criterion.
Figure 1: An example of the toe spread index used
to assess recovery.
Results
The surgical procedure and the laser, ultrasound
application was well tolerated by all rats, and no
animal died during the experiment. The study was
conducted with a total of 18 footprint images in the
different periods, preoperative, 7th, 14th, and 21st
postoperative days. They were evaluated by the toe
spread assay. These values are then analysed for their
normality using Shapiro- wilk test and equal variance
using brown-forsyth test. All the three groups passed
the test. This indicates that all three groups have a
similar baseline index and there is no potential bias
among allocated subjects.
Toe spread assay value among all the three
groups were found statistically significant comparing
its pre-OP and 21st day value. The mean difference
of group A is 8.95, group B is 5.81 and group C is
12.86. This shows that the LLLT group is very close
to normal value showing significant difference than
the other two groups. Tukey HSD test between all
three groups proved that toe spread assay measure
is statistically significant (p<0.005) between all three
groups, this indicates that even the control group has
shown improvement after 21 days of healing period.
Hence toe spread assay has been improved in all
three groups than its measure on 7th day but LLLT
group has shown little more improvement then other
two groups.
Fig 1: Representing the mean difference for Toe
Spread Assay on Pre-op, day 2, day 14 and day 21.
Discussion
Some evidence states that peripheral nerves do
in some manner respond to therapy with ultrasound,
the results of early trials were not entirely conclusive,
especially when it comes to the use of ultrasound
in individuals.11 High-intensity radiation would
prevent neuron regeneration, whereas low-intensity
therapeutic ultrasound delivered in minute doses
would promote it, according to experimental
research. This demonstrates that, as was shown in
rats, ultrasonic exposure within a relatively narrow
intensity range has a dose-related effect that is
inversely proportionate. Suganthirababu P et al.,
states that ulnar nerve radiation to low level laser
decreases the latency and increases the amplitude.13
The likelihood that therapeutic ultrasound
could have a positive impact on nerve regeneration
is what drove the current research. Since it is
not always possible to compare the outcomes of
several evaluation approaches employed by the
same author or to draw immediate inferences
from them, evaluating peripheral nerves that
are going through regeneration can be difficult.
Histologic, morphometric, and electrophysiological
investigations make up the majority of research on
peripheral nerve regeneration; while important, these
techniques don’t reveal much about the functional
recovery itself. Depending on the severity of the
nerve injury, injuries to the peripheral nerves result
in significant dysfunctions and sometimes have
lifetime consequences.15
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Suganthirababu P et al., suggest that ultrasound
therapy has a capacity to either assist or inhibit
physiological activity by changing the nerve
conduction velocity.14
In order to measure the effectiveness and speed
of nerve conduction, nerve stimulation experiments
include stimulating a nerve and monitoring the
electrical impulses that occur. The treated group
(getting ultrasound thermotherapy) would be
measured and compared to control groups as the
authors measured and analysed nerve conduction
parameters. The quantitative information and
conclusions from the nerve conduction studies
would be presented in the study’s results section.
The differences in nerve conduction parameters seen
between the treated group and the control groups
are most likely to be discussed by the authors.
The importance of the results in relation to nerve
regeneration and functional recovery following
compression neuropathy will be discussed in the
discussion section.
Conclusion
Based on our samples, data, and techniques, it was
possible to conclude that, when compared to LIPUS,
LLLT had an advantageous effect on the functional
rehabilitation of the right sciatic nerve. In the sciatic
nerve damage, the healing process was improved after
transcutaneously delivering therapeutic ultrasound
and laser to the injury site. However, when compared,
laser is more advantageous for nerve regeneration
following a severe grade of peripheral nerve injury.
Ethical clearance: The Ethics Committee,
BRULAC/SDCH/SIMATS/IAEC/01-2023/11,
Saveetha University, India, approved the
experimental study.
Funding: Self
Conflict of Interest: Nil
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