Low-Level Laser Therapy Accelerates Collateral Circulation and Enhances Microcirculation

Abstract

To evaluate the efficacy of low-level laser therapy (LLLT) on collateral circulation and microcirculation if a blood vessel is occluded.
Investigators have attempted prostaglandin and ultrasound therapy to promote improvements in the vascular bed of deprived tissue after an injury, which may lead to occlusion of the blood vessels.
Thirty-four adult rabbits were used in this study, two of them considered 0-h reading group, while the rest were divided into two equal groups, with 16 rabbits each: control and those treated with LLLT. Each rabbit underwent two surgical operations; the medial aspect of each thigh was slit, the skin incised and the femoral artery exposed and ligated. The site of the operation in the treated group was irradiated directly following the operation and for 3 d after, one session daily for 10 min/session. The laser system used was a gallium-aluminum-arsenide (Ga-Al-As) diode laser with a wavelength of 904 nm and power of 10 mW. Blood samples collected from the femoral artery above the site of the ligation were sent for examination with high-performance liquid chromatography (HPLC) to determine the levels of adenosine, growth hormone (GH) and fibroblast growth factor (FGF). Tissue specimens collected from the site of the operation, consisting of the artery and its surrounding muscle fibers, were sent for histopathological examination to determine the fiber/capillary (F/C) ratio and capillary diameter. Blood samples and tissue specimens were collected at 4, 8, 12, 16, 20, 24, 48 and 72 h postoperatively from the animals of both groups, control and treated.
Rapid increases in the level of adenosine, GH, and FGF occurred. The F/C ratio and capillary diameter peaked at 12-16 h; their levels declined gradually, reaching normal values 72 h after irradiation in the treated group. Numerous collateral blood vessels proliferated the area, with marked increases in the diameters of the original blood vessels.
The results indicated that LLLT accelerated collateral circulation and enhanced microcirculation and seemed to be unique in the normalization of the functional features of the injured area, which could lead to occlusion of the regional blood vessels.

Full text

289
Photomedicine and Laser Surgery
Volume 23, Number 3, 2005
© Mary Ann Liebert, Inc.
Pp. 289–294
Low-Level Laser Therapy Accelerates Collateral
Circulation and Enhances Microcirculation
F.R. MOHAMMED IHSAN, Ph.D.
ABSTRACT
Objective: To evaluate the efficacy of low-level laser therapy (LLLT) on collateral circulation and microcircu-
lation if a blood vessel is occluded. Background data: Investigators have attempted prostaglandin and ultra-
sound therapy to promote improvements in the vascular bed of deprived tissue after an injury, which may
lead to occlusion of the blood vessels. Materials and Methods: Thirty-four adult rabbits were used in this study,
two of them considered 0-h reading group, while the rest were divided into two equal groups, with 16 rabbits
each: control and those treated with LLLT. Each rabbit underwent two surgical operations; the medial aspect
of each thigh was slit, the skin incised and the femoral artery exposed and ligated. The site of the operation in
the treated group was irradiated directly following the operation and for 3 d after, one session daily for 10
min/session. The laser system used was a gallium-aluminum-arsenide (Ga-Al-As) diode laser with a wave-
length of 904 nm and power of 10 mW. Blood samples collected from the femoral artery above the site of the
ligation were sent for examination with high-performance liquid chromatography (HPLC) to determine the
levels of adenosine, growth hormone (GH) and fibroblast growth factor (FGF). Tissue specimens collected
from the site of the operation, consisting of the artery and its surrounding muscle fibers, were sent for
histopathological examination to determine the fiber/capillary (F/C) ratio and capillary diameter. Blood sam-
ples and tissue specimens were collected at 4, 8, 12, 16, 20, 24, 48 and 72 h postoperatively from the animals of
both groups, control and treated. Results: Rapid increases in the level of adenosine, GH, and FGF occurred.
The F/C ratio and capillary diameter peaked at 12–16 h; their levels declined gradually, reaching normal val-
ues 72 h after irradiation in the treated group. Numerous collateral blood vessels proliferated the area, with
marked increases in the diameters of the original blood vessels. Conclusions: The results indicated that LLLT
accelerated collateral circulation and enhanced microcirculation and seemed to be unique in the normaliza-
tion of the functional features of the injured area, which could lead to occlusion of the regional blood vessels.
INTRODUCTION
NEW CAPILLARIES are always formed from preexisting ones
by a process of budding. This occurs by growth during
embryonic development (angiogenesis)1and after tissue injury
through collateral circulation.2Endothelial budding is induced
by a growth factor formed by parenchymal cells in hypoxia,
possibly as a result of anaerobic glycolysis.1,2
Control of blood flow to a tissue can be divided into two
phases: (1) acute, which is achieved by rapid changes in the di-
ameter of arterioles, metarterioles, or precapillary sphincters;
and (2) long-term, which results from an increase or decrease
in the number of actual blood vessels supplying the tissue.3
During the healing process of an injured muscle, fibers begin
to increase in length and diameter many times the normal values,
which is associated with the formation of new capillaries. If new
capillaries are not formed during the regenerative process, the
capillary density will be reduced and the healing process altered.4
New capillaries are formed only if the muscle maintains a normal
blood supply, which comes from collateral circulation. Regenera-
tion of any muscle depends upon the presence of factors such as
adenosine, growth hormone (GH), and fibroblast growth factor
(FGF), also called the angiogenic factors.5
Low-level laser therapy enhances vasodilatation and prolif-
eration of the microvasculature.6Laser irradiation is also
thought to increase the level of oxygen content of tissue.7
Department of Anatomy, AL-Kindy College of Medicine, University of Baghdad, Iraq.
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290 Ihsan
MATERIALS AND METHODS
Thirty-four adult New Zealand rabbits with average body
weights of 1.5–2 kg were used in this study. They were divided
into two equal groups: a control group and a group treated with
LLLT, each group consisting of 16 rabbits, with the remaining
two rabbits considered 0 h reading group.
Each rabbit underwent two surgical operations: one in the
medial aspect of each thigh. The operations were carried out
under general anesthesia using a combination of acepromazine
malet (10 mg/kg body weight) Calmivet, 0.5 mg, Many-Venice-
70200, Lura, France), ketamine hydrochloride (10 mg/kg body
weight) (Ketallar, 50 mg/mL, Park Davis, Gwent, U.K.), and
Xylazin (5 mg/kg body weight) (Rompon, 20 mg/mL, DeH 28,
Pantex, Holland), injected intramuscularly.8The femoral artery
is the main arterial supply of the lower limb.9An incision was
made halfway down the medial aspect of the thigh to involve
the skin, superficial fascia, and the thin fascia lata. Then the
two muscles—the sartorius, anteriorly, and the adductor lounges,
posteriorly—were separated bluntly to expose the femoral
artery, which was ligated at the level of the midshaft of the
femur using 4-0 catgut (Chordasowa Resor, bills aseptica, PH-
Eue, Berlin, Germany).
Blood samples were collected from the femoral artery above
the ligation and examined with high-performance liquid chro-
matography (HPLC, Stamps. Analytical Laboratory Instru-
ment, Japan) to determine the levels of adenosine, according to
the United States Pharmacopoeia,10 GH, according to Smith
and Lee,11 and FGF levels, according to the British Pharma-
copoeia.12
Tissue specimens were taken from the site of ligation
(transverse sections), which included the artery and its sur-
rounding muscle fibers. The specimens were fixed and stained
with hematoxyline and eosin (H&E) stain. The specimens were
examined histopathologically to determine the fiber/capil-
lary (F/C) ratio and the capillary’s diameter, according to
Tomanek et al.13,14 The capillary diameter (7–10 capillaries)
was measured during each period, depending on the degree
of healing, using a micrometer (Micro, Bausch & Lomb,
United States).
Blood samples and tissue specimens at 0-h were collected
from the two rabbits, which were considered the 0-hour read-
ing group. Two rabbits from each group, the control and laser-
treated, were identified for collection of blood samples, and
tissue specimens were measured at 4, 8, 12, 16, 20, 24, 48, and
72 h postoperatively. Those in the treated group were irradi-
ated immediately postoperatively and for 3 d thereafter. They
received one session daily for 10 min/session using a gallium-
aluminum-arsenide (Ga-Al-As) diode laser system (Russian-
Palisb, Moskovesky Polisib, Moscow, Russia) with a 904-nm
wavelength at 10 mW. The laser beam was applied directly on
the site of the operation.
The values obtained from blood samples and tissue speci-
men examinations were estimated statistically using ANOVA
and LSD to compare the responses of both the control and
laser-treated groups throughout the entire period of the study.15
RESULTS
In the treated group, HPLC showed a rapid increase in
the levels of adenosine, GH, and FGF, reaching peak at
12–16 h and gradually dropping to normal values by 72 h
(Tables 1–3).
The histopathological examination indicated an increase in
the F/C ratio and the capillary diameter in the treated group,
which corrolated with the HPLC readings (Tables 4 and 5).
Statistical analysis revealed a very highly significant (p<
0.001) response in the animals of the treated group from the
occlusion of the femoral artery, beginning at the fourth hour
after irradiation and peaking at the twelfth hour. The normal-
ization of the injured area and whole body function com-
menced early in a very highly significant manner (p< 0.001),
beginning at the twentieth hour and continuing until the end of
the study. The results obtained from the treated group were
very highly significant (p< 0.001), when compared with those
of the control group for the same periods.
The level of adenosine and GH and F/C ratio reached peak
12 h after irradiation with the laser (p< 0.001), while in the
TABLE 1. MEAN AND STANDARD DEVIATIONS OF ADENOSINE CONCENTRATION
FOR CONTROL AND LASER-TREATED GROUPS
Time (h) Control (nM/mL) Laser (nM/mL) Level of significance
0 2.475 ± 0.04 2.475 ± 0.43 NS
4 3.76 ± 0.22 5.520 ± 0.03 **
8 5.435 ± 0.04 8.910 ± 0.04 **
12 5.518 ± 0.15 9.760 ± 0.02 **
16 5.435 ± 0.05 9.705 ± 0.01 **
20 9.166 ± 0.27 7.010 ± 0.02 **
24 9.652 ± 0.39 4.310 ± 0.06 ***
48 7.547 ± 0.34 3.180 ± 0.03 **
72 7.496 ± 0.24 2.480 ± 0.034 **
NS, not significant (p> 0.05); *, significant (p< 0.05); **, highly significant (p< 0.01); ***, very highly
significant (p< 0.01).
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LLLT Enhances Collateral Circulation and Microcirculation 291
TABLE 2. MEAN AND STANDARD DEVIATIONS OF GROWTH HORMONE CONCENTRATIONS
FOR CONTROL AND LASER-TREATED GROUPS
Time (h) Control (ng/mL) Laser (ng/mL) Level of significance
0 0.96 ± 0.72 0.96 ± 0.8 NS
4 1.68 ± 0.16 2.80 ± 0.04 *
8 2.74 ± 0.73 3.96 ± 0.81 **
12 3.51 ± 0.10 4.99 ± 0.84 **
16 3.76 ± 0.09 4.81 ± 0.07 **
20 4.65 ± 0.07 3.10 ± 0.73 **
24 4.91 ± 0.08 2.56 ± 0.74 **
48 4.82 ± 0.07 1.73 ± 0.07 **
72 4.44 ± 0.08 0.98 ± 0.83 **
NS, not significant (p> 0.05); *, significant (p< 0.05); **, highly significant (p< 0.01); ***, very highly
significant (p< 0.01).
TABLE 3. MEAN AND STANDARD DEVIATIONS OF FIBROBLAST GROWTH FACTOR CONCENTRATIONS
FOR CONTROL AND LASER-TREATED GROUPS
Time (h) Control (ng/mL) Laser (ng/mL) Level of significance
0 0.0031 ± 0.0017 0.0031 ± 0.001 NS
4 0.0051 ± 0.001 0.0140 ± 0.002 NS
8 0.0077 ± 0.001 0.0320 ± 0.002 **
12 0.0096 ± 0.0014 0.0486 ± 0.006 **
16 0.0101 ± 0.0014 0.0398 ± 0.003 **
20 0.0234 ± 0.0017 0.0291 ± 0.0014 NS
24 0.0288 ± 0.003 0.0120 ± 0.001 NS
48 0.0310 ± 0.002 0.0093 ± 0.011 *
72 0.0449 ± 0.001 0.0042 ± 0.001 *
NS, not significant (p> 0.05); *, significant (p< 0.05); **, highly significant (p< 0.01); ***, very highly
significant (p< 0.01).
TABLE 4. MEAN AND STANDARD DEVIATIONS OF FIBER/CAPILLARY RATIO FOR CONTROL
AND LASER-TREATED GROUPS
Time (h) Control (µm) Laser (µm) Level of significance
0 1:0.38 ± 0.01 1:0.38 ± 0.01 NS
4 1:0.41 ± 0.05 1:0.9 ± 0.19 **
8 1:1.025 ± 0.32 1:1.5 ± 0.11 **
12 1:1.66 ± 0.08 1:2.2 ± 0.37 **
16 1:1.65 ± 0.07 1:1.2 ± 0.67 **
20 1:1.1 ± 0.08 1:0.9 ± 0.2 *
24 1:0.78 ± 0.07 1:0.5 ± 0.18 *
48 1:0.75 ± 0.07 1:0.4 ± 0.19 *
72 1:0.58 ± 0.101 1:0.35 ± 0.06 *
NS, not significant (p> 0.05); *, significant (p< 0.05); **, highly significant (p< 0.01); ***, very highly
significant (p< 0.01).
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292 Ihsan
control group they peaked 24 h postoperatively. The level of
the FGF was highly significant (p< 0.01), reaching a peak at
4–12 h in the treated group. Normalization began 16 h after ir-
radiation in the treated group and continued until the end of the
experiment (p< 0.01). They also revealed highly significant
variations (p< 0.01) when compared with those obtained from
the control group for the same periods. There was a rapid in-
crease in the capillary diameter 4–12 h after irradiation, and
normalization began at 16 h and continued until the end of the
experiment in the treated group. When these results were com-
pared with those obtained from the animals of the control
group for the same periods, they showed significant variations
(p< 0.5) in the diameter of the capillaries to the end of the
experiment.
DISCUSSION
The factors that cause a large artery to develop into a more
or less constant shape are not completely understood. Probably
in the earliest embryonic stages, the formation of the vessels
takes place through heredity. Later, the shape and growth of the
blood vessels are determined by local chemical and hemody-
namic factors.16 Many authors have proved increased vascular-
ization of the sites with LLLT. This result has been shown to be
a laser-specific reaction.6
Growth hormone stimulates G-protein, which facilitates
cyclic adenosine monophosphate (cAMP). The latter, in turn,
stimulates cyclic deoxyribonucleic acid (cDNA); this prod-
uct will stimulate the messenger ribonucleic acid (mRNA),
which enhances and increases protein synthesis. Prosta-
glandins exaggerate the stimulatory effect of GH, cAMP, and
proteinkinase.5
FGF stimulates phosphodiesterase that converts pentos
phosphate shunt to yield nicotinamide diphosphate (NADPH).5
This compound converts to nicotinamide dihydrogen (NAD)
and adenosine triphosphate (ATP). The latter, in turn, breaks
down to adenosine diphosphate (ADP) and a phosphorous
molecule, while the oxygen molecules convert to free radi-
cals. Prostaglandins enhance the stimulatory effect of FGF.17
Low-level lasers also activate ATP, ATPase, and the conver-
sion of adenosine triphosphate to adenosine. Adenosine stimu-
lates the conversion of cAMP to nitric oxide (NO) or the
vascular endothelial growth factor (VEGF).18
Adenosine, GH, FGE, and VEGF are angiogenic factors and
promote new vessel growth in the same manner.3Endothelial
budding is induced by the angiogenic factors, which are se-
creted by the parenchymal cells in hypoxic states, possibly as a
product of anaerobic glycolysis formed at the site of these cells
and diffused in all directions.1,2
The success of collateral circulation and rapid appearance
of the microcirculation as a part of the compensatory mecha-
nism of the body against the sudden occlusion of the femoral
artery may be due to the early anastamosis between the per-
forating branches of the profunda femoris artery with the ar-
ticular and muscular branches of the same artery with those
of the popliteal artery when the femoral artery is ligated
above the adductor canal.9Thus, we can explain the rapid in-
crease in the number and diameter of the capillaries within
the first hours until the peak at the twelfth hour after irradia-
tion with low-level lasers and the subsequent decrease to
near-normal values.
The results of the present study agreed with those obtained
by Maegawa et al.,19 who investigated the effect of low-level
lasers on the rat mesenteric microcirculation in vivo. They
provide a potent dilation of the arterioles irradiated with
laser, followed by a marked increase in the arteriolar blood
flow.
CONCLUSIONS
Low-level laser emission increased tissue oxygenation, mor-
phofunctional activity, and substantial expansion of the micro-
circulatory bed. They, in turn, accelerated the restoration of
functions, stimulation of adaptational ability, and stabilization
of the hormonal status.
TABLE 5. MEAN AND STANDARD DEVIATIONS OF CAPILLARY DIAMETER FOR CONTROL
AND LASER-TREATED GROUPS
Time (h) Control (µm) Laser (µm) Level of significance
0 0.44 ± 0.045 0.44 ± 0.037 NS
4 0.46 ± 0.063 0.68 ± 0.056 NS
8 0.5 ± 0.86 0.78 ± 0.056 NS
12 0.53 ± 0.063 0.95 ± 0.031 *
16 0.59 ± 0.576 0.94 ± 0.054 *
20 0.65 ± 0.089 0.83 ± 0.067 NS
24 0.68 ± 0.067 0.72 ± 0.077 NS
48 0.64 ± 0.091 0.63 ± 0.161 NS
72 0.62 ± 0.092 0.49 ± 0.06 NS
NS, not significant (p> 0.05); *, significant (p< 0.05); **, highly significant (p< 0.01); ***, very highly
significant (p< 0.01).
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LLLT Enhances Collateral Circulation and Microcirculation 293
FIG. 1. Gutters of the blood vessels and the surrounding muscle fibers at the time of operation (0 time). (Left, H&E 4.5;
right, H&E 10.)
FIG. 2. Highly vascularized area in the treated group 12 h after irradiation with laser (left, H&E 10), with significant increase
in the diameter of the blood vessels (right, H&E 20).
FIG. 3. Beginning of the normalization of the blood vessels and surrounding muscle fibers in the animals of the treated group
16 h after irradiation (left, H&E 20). Appearance of well-developed arteries in the animals of the treated group 72 h after irra-
diation (right, H&E 40).
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294 Ihsan
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Address reprint requests to:
FR. Mohammed Ihsan, Ph.D.
Department of Anatomy
Al-Kindy College of Medicine
University of Baghdad
Baghdad, Iraq
13987C08.PGS 6/6/05 4:10 PM Page 294

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