الفهرس 
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Abstract
Sono-photodynamic therapy (SPDT) is a safe, non-toxic and non-invasive way of
destroying cancer cells, as well as enhancing immune protective function. Both sono-dynamic therapy (SDT) and photo-dynamic therapy (PDT) have been used for years as
separate processes. Photodynamic therapy (PDT) involves three individually non-toxic
components that are combined to induce cellular andtissue effects. The first component is
a photo-sensitiser molecule that localizes preferentially in certain kinds of cells and/or
tissues. The second component involves the administration of lightof a specific
wavelength. None of these factors is harmful by itself but in the presence ofoxygen.The
third component, produces reactive oxygen species (ROS) which are cytotoxic agents that
can inactivate tumor cells. This treatment modalityshows a dual selectivity that is
produced by both a preferential uptake of the photosensitiser by the diseased tissue and its
ability to confine activation of the photosensitiser by restricting the illumination to that
specific area.
Aim of the work
The present work aims to cure Ehrlich ascites carcinoma tumor implanted to group of
mice as an experimental animals using a sonophotodynamic modality in combination with
chlorophyll as a sonophoto sensitizer drug. Two sources of energy were used; namely
infrared laser at three frequency levels (1000, 4000, and 7000 Hz) with power density 16.8
mW/cm
2
and Ultrasound (pulsed and continuous wave mode) at power density 3 W/cm
2
for 1 min.
Material
A total of 140 male Swiss albino mice Tumor bearinganimals were divided into sub
groups each of 10 mice as follows:
Control mice:10 mice served as control group, i.e., free of tumor.Tumor- bearing mice
were divided into the following groups:
group I:(20 mice)
a) 10 mice: tumor bearing mice without treatment.
b) 10 mice: Tumor bearing mice treated with (Chlorophyll) as sonophotosensitizer only.
group II:(20 mice, laser irradiated group)
a) 10 mice: Exposed to Infra-Red Laser, 4000Hz, for3 minutes.
b) 10 mice: Exposed to Infra-Red Laser, 7000Hz, for3 minutes.
group III:(20 mice, ultrasound group)
a) 10 mice: Exposed to pulsed ultrasound at power density of 3W/cm
2
for 3 minutes.
b) 10 mice: Exposed to continuous ultrasound at power density of 3W/cm
2
for 3 minutes.
group IV:(20 mice, Chlorophyll, laser group) The mice of this group were injected
intraperitoneally (IP) with (Chlorophyll), then the tumor site were irradiated to laser light at
same conditions of group II.
group V:(20 mice, Chlorophyll, ultrasound group)The mice of this group were injected
(IP) with (Chlorophyll), then were divided into 2 sub-groups. The tumor site was irradiated
to ultrasound at same conditions of group III.
group VI:(30 mice, combined treatment groups)
a) 10 mice: The tumor site was irradiated to laser light for 3 minutes, followed by
ultrasound 3W for 3 minutes.
b) 10 mice: Injected (IP) with (Chlorophyll). The tumor site was irradiated to laser light for
3 min, followed by ultrasound 3W for 3 minutes.
c) 10 mice: Injected (IP) with (Chlorophyll + Vitamin B
12
). The tumor site was irradiated
to laser light for 3 minutes, followed by ultrasound 3W for 3 minutes.
The treatment effects evaluation:
• The dimensions of the tumor, e.g., the length, the width and the height of were
measured using a slide digital caliper every day before any treatment modality. The
tumor volume was calculated.
• Tumor mass inhibition ratio and tumor volume growthratio were also calculated.
• Biochemical examinations were appliedto detect serum levels of ALT, AST, urea
and creatinine to detect the effect of CHLinjectionon liver and kidney functions.
• The concentrations of selenium (Se), iron (Fe), nickel (Ni), chromium (Cr),
cadmium (Cd), lead (Pb), zinc (Zn) and copper (Cu) were estimated using Atomic
absorption spectrophotometry.
• Activities of some antioxidants were measured, namely; glutathione-S-transferase
activity (GST), super oxide dismutase activity (SOD), glutathione reductase
activity (GR), catalase activity (Cat), total antioxidant activity (TAC) and
malondialdehyde (MDA).
• Evaluating thehistopathologicaland ultra-structuralchanges in the tumor tissues
following the different treatment methodsusing Hematoxylin and Eosin (H&E)
stainusing light microscope and transmission electron microscope (TEM).
Results of thestudy:
• The injection of the photosensitizer (chlorophyll) has no effect on the tumor
volume.
• The effect of exposing the tumor to IR laser as a photodynamic therapy increased
with increasing the laser energy resulted in decreasing the tumor volume, tumor
growth rate and inhibition ratio. These effects were observed either on using
infrared laser alone (with its two frequencies) or in the presence of the CHL.
• The inhibition in thetumor volume has minimum valueon using 7000 Hz IR laser
in the presence of the CHL.
• The effect of exposure to continuous ultrasound wave was more than that in case of
using pulsed ultrasound wavebut with high mortalityrate in mice as a small
models. Similar variations occurred in case of using ultrasound exposure only or in
the presence of the CHL, with minimum effect occurred on using ultrasound in the
presence of the CHL.
• Combined treatment of IR laser at 7000 Hz and pulsed ultrasound wave at 3W/cm
2
in the presence ofchlorophyll was more effective than either IR laser or ultrasound
alone.
• The presence of Vit.B12
have no significant effect in the combined treatment.
• The histopathological and ultra-structural changes reflected inertial ultra-structural
destruction in nuclear and cell organelles.
• In the group of mice carrying the tumor only, a significant increase in the levels of
MDA as compared to the control group of animals. The combination of
photodynamic andsonodynamic therapy in the presences of chlorophyll
decreasedsignificantly the levels of MDA.
• In all Ehrlich bearing mice groups decreased activities of enzymatic antioxidants
(SOD, CAT, GR, GST and TAC) in comparison with normal group were observed.
• A significant increase in the enzymatic and non-enzymatic antioxidant guard were
observed in the groupssubjected to combination of photodynamic andsonodynamic
therapy in the presences of chlorophyll.
• A statistically significant elevation of heavy metals and trace elements(lead,
cadmium, chromium, nickel and iron) concentrations was detected in serum of
mice group bearing the tumor compared to the control group.
• A significant decrease in theheavy metals and traceelements was observed in
thegroup of mice subjected tocombination of photodynamic andsonodynamic
therapy in the presence of chlorophyll.
• It was observed that treatment with CHLameliorated the levels of serum creatinine
and urea which is an indication of renal protection. This also confirms the
protective role of CHLagainst renal toxicity. Also treatment with CHLprotected
against increase in serum levels of ALT, AST, and GGT, which is an indication of
hepatoprotection by CHL. This also confirms the protective role of CHL against
hepatotoxicity.
• The histological evaluation revealed that all tumors from the group of mice bearing
the tumor included highly malignant cells and none of the tumors showed necrosis.
Tumors excised from animals receiving treatment (CHL)showed significant areas
of necrosis compared to groups without sensitizer.
• In the group of animals subjected to combination ofphotodynamic
andsonodynamic therapy in the presences of chlorophyll, large foci distinct
necrosis areas were appeared.
Conclusion:
• Chlorophyll apotential photosensitizer and sonosensitizer for photodynamic or
sonodynamic treatment of Ehrlich ascites tumor tissue.
• Infra-red laser in combintion with ultrasound in the presence ofCHL has a potential
antitumor effect.
• Ultrastructure changes play important roles in inhibiting tumor growth and even
inducing cell death, which might be attributed to Photo or sono-chemical activation
mechanism.
• B12
has no effect in the combination of photodynamic andsonodynamic therapy in
the presences of chlorophyll as a sensitizer.