Ozone Therapy on Cerebral Blood Flow: A Preliminary Report

Ozone Therapy on Cerebral Blood Flow: A Preliminary Report
Bernardino Clavo1,2,7, Luis Catalá3,7, Juan L. Pérez2,4,7, Victor Rodríguez5 and
Francisco Robaina2,6,7 Departments of 1Radiation Oncology and 2Research Unit, 3Radiology, 4Medical Physics and 6Chronic Pain Unit, Dr Negrín Hospital, 5La Paterna Medical Center and 7Canary Islands Institute for Cancer Research (ICIC), Las Palmas (Canary Islands), Spain

Introduction
Cerebral low perfusion syndromes have significant clinical
and social repercussions. An important field in neurological
research includes the search for more effective drugs and other
methods in order to ameliorate this problem. Ozone therapy is
a non-conventional therapy that has been used for several years
in the treatment of ischemic disorders, particularly of the
lower limbs (1–3). However, to date, very few studies have
systematically evaluated blood flow changes resulting from
ozone therapy.
With regard to this, the effect of ozone therapy on the blood
flow in the middle cerebral artery (MCA) and the common
carotid artery (CCA) was investigated in the current study.
Subjects and Methods Patients In this study, the blood flow in 28 arteries (14 MCA and
14 CCA) was evaluated in 7 subjects—5 patients and 2 healthy
volunteers. The subjects were from our university hospital.
The patients who underwent elective ozone therapy, which was
unrelated to the treatment of their cerebral vascular diseases,
were from the Radiation Oncology department. Their scheduled
medication was not modified during the study period. The volunteers
were members of the clinical staff of the departments
Advance Access Publication 6 October 2004 eCAM 2004;1(3)315–319
doi:10.1093/ecam/neh039
© 2004, the authors
Evidence-based Complementary and Alternative Medicine, Vol. 1, Issue 3 © Oxford University Press 2004; all rights reserved
Original Article
Ozone Therapy on Cerebral Blood Flow: A Preliminary Report
Bernardino Clavo1,2,7, Luis Catalá3,7, Juan L. Pérez2,4,7, Victor Rodríguez5 and
Francisco Robaina2,6,7
Departments of 1Radiation Oncology and 2Research Unit, 3Radiology, 4Medical Physics and 6Chronic Pain Unit,
Dr Negrín Hospital, 5La Paterna Medical Center and 7Canary Islands Institute for Cancer Research (ICIC),
Las Palmas (Canary Islands), Spain
Ozone therapy is currently being used in the treatment of ischemic disorders, but the underlying mechanisms
that result in successful treatment are not well known. This study assesses the effect of ozone
therapy on the blood flow in the middle cerebral and common carotid arteries. Seven subjects were
recruited for the therapy that was performed by transfusing ozone-enriched autologous blood on 3 alternate
days over 1 week. Blood flow quantification in the common carotid artery (n
14) was performed
using color Doppler. Systolic and diastolic velocities in the middle cerebral artery (n
14) were estimated
using transcranial Doppler. Ultrasound assessments were conducted at the following three time
points: 1) basal (before ozone therapy), 2) after session #3 and 3) 1 week after session #3. The common
carotid blood flow had increased by 75% in relation to the baseline after session #3 (P  0.001) and by
29% 1 week later (P
0.039). In the middle cerebral artery, the systolic velocity had increased by 22%
after session #3 (P
0.001) and by 15% 1 week later (P
0.035), whereas the diastolic velocity had
increased by 33% after session #3 (P  0.001) and by 18% 1 week later (P
0.023). This preliminary
Doppler study supports the clinical experience of achieving improvement by using ozone therapy in
peripheral ischemic syndromes. Its potential use as a complementary treatment in cerebral low perfusion
syndromes merits further clinical evaluation.
Keywords: color Doppler – ischemia – low perfusion – transcranial Doppler
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derivative work this must be clearly indicated.
For reprints and all correspondence: Bernardino Clavo, MD, Department of
Radiation Oncology and Research Unit, Dr Negrín Hospital, C/ Barranco la
Ballena s/n, 35020 Las Palmas (Canary Islands), Spain. Fax: 34 928
449127; Tel: 34 928 450284. E-mail: bernardinoclavo@terra.es
involved in the investigation. The study included 5 males and
2 females with a mean age of 58 years (range, 34–78).
Informed consent was obtained from all the participants prior
to inclusion in the study. The study was approved by the
Institutional Ethical Committee. Table 1 summarizes the
details of the subjects that participated in this study.
Ozone Therapy
Ozone therapy was administered by autologous blood transfusion
on 3 alternate days over 1 week. The procedure involved
the collection of 200 ml venous blood into a blood bag containing
heparin (25 IU/ml) and CaCl2 (5 mM). The O3/O2 gas
mixture was prepared from clinical-grade O2 using the OZON
2000 medical device (Zotzmann  Stahl GmbH, Plüderhausen,
Germany). The blood was mixed with 200 ml of O3/O2 gas
mixture at a concentration of 60
g/ml in a sterile single-use
300 ml container. Subsequently, the blood was slowly
re-introduced into the patient via the antecubital vein, after
being passed through a sterile 0.20
m filter. The blood
remained outside the body for approximately 15–30 min, and
no adverse reactions were observed.
Doppler studies were conducted on the following three
occasions: 1) before session #1; 2) after session #3; and 3)
1 week after session #3.
Transcranial Doppler Velocimetry
Systolic and diastolic velocities (in cm/s) were measured in the
MCA by the transtemporal approach using a transcranial
Doppler (TCD) with a 2 MHz probe from an Angiodine-2
Fluo-Link 300® device. The patient was alert, relaxed and
seated when the absence of stenoses was confirmed. The
Doppler insonation angle was 60.
Common Carotid Blood Flow Quantification
Blood flow quantification of CCA was performed using
a color Doppler, Philips Ultrasound P-800 unit®, with timedomain
processing. This technique simultaneously evaluates
the velocity and the vessel diameter, and the data is presented
in terms of ml/min. The usefulness and validity of this technique
has been previously described (4,5). The patient was
alert, relaxed and in the supine position when the absence of
significant stenoses in the extracranial carotid arteries was
confirmed. A 7.5 MHz linear high-definition probe with a
Doppler insonation angle of 60 was used. We obtained
information regarding the volume of blood flow (in ml/min) in
both CCAs at 2 cm prior to the carotid bifurcation.
All ultrasound studies were performed bilaterally by the
same radiologist in order to minimize interobserver variability
(6). When an optimal image of stable blood flow was obtained,
recordings over at least three cardiac cycles were made. This
was repeated at least three times in order to preclude operatorinduced
or technical inaccuracies. The median values that
were obtained were used in the statistical analyses.
Neither blood pressure nor hemoglobin levels were measured.
Statistical Analysis
The SPSS 7.0 for Windows software package (SPSS-Ibérica,
Madrid, Spain) was used throughout the study. The normality
of distribution of data was assessed by the Kolmogorov–
Smirnov test. Two-sided tests were applied. The data are
expressed as mean  SD. The paired t-test was used to compare
differences between the baseline and the two time-point
measurements following the ozone therapy. Linear correlation
was assessed by the Pearson’s r test. The differences were
considered to be significant when P  0.05.
316 Ozone therapy on cerebral blood flow
Table 1. Patients and control subjects included in the study
Patient Age (years) Characteristics
#1 67 aComplementary treatment during radiochemotherapy for advanced carcinoma of hypopharynx.
bArterial hypertension under drug treatment and hyperglycemia under dietary treatment.
#2 74 aComplementary treatment during radiochemotherapy for advanced carcinoma of base of tongue.
bChronic obstructive bronchitis (COB) under treatment with bronchodilator inhalers.
#3 63 aComplementary treatment during radiochemotherapy for advanced carcinoma of supraglottis.
bHyperuricemia treated with allopurinol. Multiple sclerosis treated with baclofen.
#4 51 aRadiation-induced necrosis of thyroid cartilage (radiotherapy was administered for carcinoma of glottis several years ago).
bTreatment with corticosteroids.
#5 78 aChronic ulceration with calcaneous exposure and transplant failure.
bInsulin-dependent diabetes, arterial hypertension under drug treatment. Stroke 1 year ago. Duodenal ulcers.
#6 43 Healthy subject.
#7 34 Healthy subject.
aReason for ozone therapy. bConcomitant diseases or treatments (no changes were made in the medications during the period of Doppler evaluation). The study
was planned with three ozone therapy sessions to evaluate the initial effects under the same conditions. Patient #1, #2 and #3 (cancer patients) were required to
commence their scheduled radiochemotherapy after session #3; therefore, ethical considerations precluded delay in the cancer treatment. Ozone therapy was
continued during the radiochemotherapy; however, radiotherapy of the cervical and carotid areas altered the subsequent Doppler evaluations. Patient #4 suffered
from hemorrhage of the larynx. Ozone therapy was stopped after session #3 to enable the patient to undergo surgery. The usual complications associated with the
surgical treatment of this radiation-induced necrosis were absent. Patient #5 who suffered from several vascular diseases was treated with systemic and local
ozone therapy for a chronic wound. Patient #6 and #7 (healthy subjects recruited from among the hospital staff) also received 3 sessions of ozone therapy to
evaluate the Doppler Effect. Further sessions were neither scheduled nor administered.
eCAM 2004;1(3) 317
Results
Transcranial Doppler Velocimetry
The baseline systolic velocity in MCA was 90.9  6.1 cm/s.
After session #3, it increased to 111  7.3 cm/s (increase 22%,
P
0.001), and 1 week later, it was 104.3  8 cm/s (increase
15%, P
0.035). The baseline diastolic velocity in MCA was
41.1  4.4 cm/s. After session #3, it increased to 54.6  4.6 cm/s
(increase 33%, P  0.001), and 1 week later, it was 48.6 
5 cm/s (increase 18%, P
0.023) (Fig. 1).
Common Carotid Blood Flow Quantification
The baseline CCA blood flow was 233  19 ml/min. After
session #3, it increased to 407  38 ml/min (increase 75%,
P  0.001), and 1 week later, it was 301  22 ml/min
(increase 29%, P
0.039) (Fig. 2).
The baseline CCA blood flow directly correlated with the
MCA diastolic velocity (r
0.557; P
0.039) and inversely
correlated with age (r
0.825; P  0.001) (Fig. 3). The percentage
increase in CCA blood flow 1 week after session #3
was directly correlated with age (r
0.735; P
0.004)
(Fig. 4) and inversely correlated with the initial values of the
CCA blood flow (r
0.691; P
0.009). In older patients, the
increase in CCA blood flow was higher and that in basal perfusion
was lower (Fig. 5) (Note: in Figs 4 and 5, the Doppler
data for the left arteries of one patient 1 week after session #3
were not available due to technical reasons).
Discussion
Although biomedical applications of ozone therapy can be
traced back to the end of the 19th century, numerous aspects of
the effects of the therapy remain unexplored.
The airways are precluded in this therapy, which uses ozoneenriched
autologous blood transfusion; therefore, lung toxicity
resulting from oxidative stress is avoided. Ozone, per se, does
not enter the organism; the effects that are observed are mediated
0
20
40
60
80
100
120
pre-1 post-3 1 week
* *
0
10
20
30
40
50
60
70
pre-1 post-3 1 week
*
*
MCA diastolic velocity - cm/s
MCA systolic velocity - cm/s
Figure 1. Transcranial Doppler during ozone therapy. Left. Diastolic velocity (in cm/s) in the middle cerebral artery (MCA) increased by 33% at the end of session
#3 (P  0.001), and an 18% increase persisted for 1 week after session #3 (P
0.023). Right. Systolic velocity in MCA increased by 22% at the end of session #3
(P
0.001), and a 15% increase persisted for 1 week after session #3 (P
0.035). The error bars are the 95% confidence intervals. Significant differences
(P  0.05) are indicated with an asterisk (*).
0
50
100
150
200
250
300
350
400
450
pre-1 post-3 1 week
*
*
CCA blood flow - ml / min
Figure 2. Carotid blood flow during ozone therapy. Blood flow quantification
(in ml/min) in the common carotid artery (CCA) increased by 75% at the end
of session #3 (P  0.001), and a 29% increase persisted for 1 week after
session #3 (P
0.039). The error bars are the 95% confidence intervals.
Significant differences (P  0.05) are indicated with an asterisk (*).
0
10
20
30
40
50
60
70
80
90
100 150 200 250 300 350
Basal CCA blood flow - ml/min
Age
Figure 3. Relationship between baseline blood flow and age. Baseline values
of the common carotid artery (CCA) blood flow were inversely correlated with
the age of the patients (r
0.825; P  0.001). A lower blood flow was
observed in older patients.
by the rapid oxidation of certain substances in the blood in the
transfusion recipient. In appropriate concentrations, this can
up-regulate the synthesis of antioxidants in blood (7). This
property has been very actively investigated with respect to the
protection against free radical damage associated with heart
(8), kidney (9) and liver (10) disorders. The mechanisms
proposed to explain the vascular effects include the liberation
of vasoactive substance as well as the improvement in erythrocyte
flexibility and blood rheology (1,11,12).
Several studies that included control subjects have indicated
that when ozone-free oxygen is used, the beneficial biochemical
(7,10) and rheological (1) responses are not observed. The
changes in the MCA and/or CCA blood flow occurring during
ozone therapy were assessed in the present study that did not
include non-ozonized blood transfusion and each patient was
his/her own control.
As indicated by the CCA measurements, the increase in
diastolic velocity in the MCA is compatible with a decrease
in vascular resistance, a rheological improvement (1,12) and
an overall increase in blood flow. The inverse correlation
between the percentage increase in CCA blood flow and the
initial values is compatible with a microvascular redistribution
resulting in better oxygenation in tissues with poor blood supply.
This was tentatively demonstrated in our previous studies
by the direct measurement of muscle and tumor oxygenation
using polarographic electrodes (13,14).
These rheological and vascular effects suggest that coadjuvant
ozone therapy could decrease the vasoconstriction that is secondary
to hyperoxia. Techniques such as carbogen breathing or
hyperbaric chambers are used to increase the amount of O2 dissolved
in arterial blood. However, when prolonged for 15–30
min, these therapies can lead to an increase in peripheral vascular
resistance along with a generalized vasoconstriction in most
organs (15). Decreased cerebral blood flow secondary to hyperoxia
has indeed been documented in humans by transcranial
Doppler (16) and magnetic resonance (17) studies.
The above-mentioned effects of ozone therapy and data from
the present study, especially the potentially greater effect in
older patients or in those with lower initial blood flow, augur
well for its use in cerebral low perfusion syndromes and stroke.
This is further supported by the clinical experience gained in a
study that assessed 150 patients with ischemic cerebrovascular
disease treated with prolonged ozone therapy (18).
The present Doppler study was planned with only three
ozone therapy sessions for several reasons. Firstly, we wanted
to evaluate the effect of ozone therapy and to observe whether
the effect could be maintained for a prolonged period, which
has been suggested by the clinical experience gained from its
use in sessions widely separated over several days. Hence, we
decided to perform the third session approximately 1 week
later without any intervening sessions. Secondly, we wanted to
administer the same number of sessions to all the patients in
the study. However, some of them were cancer patients who
needed to commence their scheduled radiochemotherapy.
Therefore, in order to avoid interference with the scheduled
radiochemotherapy, the present ozone study was performed
during the period when oncologic staging and planning of the
radiotherapy were carried out. Hence, the number of ozone
therapy sessions for Doppler evaluation was less than that
considered necessary for a full-fledged ozone therapy, which
usually lasts for several weeks or even months. The Doppler
Effect after several additional sessions could indeed be higher
than that currently observed. Data on the optimal separation
between the ozone therapy sessions are not currently available.
Further, the schedule could vary depending on the desired
clinical effect (antioxidant, enhancing the immune or vascular
system, etc.). Nevertheless, the current study supports the
clinical experience gained in the treatment of vascular disorders,
318 Ozone therapy on cerebral blood flow
0
50
100
150
200
250
300
350
0 10 20 30 40 50 60 70 80
Age
% increase in CCA blood flow
1 week after ozone therapy
Figure 4. Relationship between age and blood flow increase post-ozone
therapy. The percentage increase in CCA blood flow 1 week after session #3
was directly correlated with age (r
0.735; P
0.004). A higher increase
was observed after ozone therapy in older patients.
0
50
100
150
200
250
300
350
100 150 200 250 300 350
Basal CCA blood flow - ml/min
% increase in CCA blood flow
1 week after ozone therapy
Figure 5. Relationship between the baseline blood flow and its increase post
ozone therapy. The correlation in CCA blood flow between baseline values
and the percentage increase 1 week after session #3 was highly significant
(r
0.691; P
0.009), i.e., there is a higher percentage increase in CCA
corresponding to a lower initial blood flow. Note: the percentages under 100%
indicate a decrease in blood flow at this time point.
eCAM 2004;1(3) 319
employing widely separated sessions over extended periods
(2,3). Two or three applications per week appear to be sufficient
in providing significant vascular improvement. However,
changes observed over a mere 1 or 2 weeks are usually not
sufficient to improve chronic clinical conditions. The current
findings regarding a residual effect, which is still significantly
elevated over baseline 1 week after the last session, support
our postulation that one or two additional sessions per week
can be effective during the initial maintenance period. The
mode and timing of administration of additional sessions over
a period of months need to be explored for the optimization of
the sessions.
In the course of this study, our hospital facilities were transferred
to a different location in our city, and we were unable to
conduct further Doppler studies using the same equipment.
Therefore, we decided to increase the study sample by including
two healthy subjects from our hospital. We could not evaluate
the differences between patients and healthy subjects due to
the scarcity of patients. Only patient #5 had suffered a
CVA/stroke that may modify the Doppler evaluation in the
carotid and middle cerebral arteries. However, patients with
localized tumors do not appear to have a systemic vascular
alteration or an altered vascular response. Therefore, we
assumed that the effect observed in these arteries is a general
effect, which does not differ from that observed in the healthy
subjects or the patients that were studied.
Further studies, which include new technologies such as
interstitial multichannel laser Doppler used to quantify fluctuations
in microvascular perfusion during ozone therapy, are in
progress in order to ascertain some of the remaining doubts
regarding the efficacy of ozone therapy.
In conclusion, this preliminary Doppler study demonstrates,
albeit in a small number of subjects, that ozone therapy
increases blood flow in CCA and MCA with a prolonged
effect such that it can be very easily assessed by TCD and
carotid ultrasound. These data support the clinical experience
of achieving improvement using ozone therapy in peripheral
ischemic syndromes. Its potential use as a complementary
treatment in cerebral low perfusion syndromes warrants
further clinical investigation.
Acknowledgements
The study was supported in part by a grant (FUNCIS 98–31)
from the Health and Research Foundation of the Autonomous
Government of the Canary Islands, Spain.
We wish to thank Dr R. Reyes (Department of Interventional
and Vascular Radiology) and Dr G. Rovira-Dupláa (Ozone
therapy Unit of the Quirón Clinic, Barcelona, Spain) for their
valuable advice in conducting this study. We also thank
R. Martin-Oliva (Head of Department of Medical Physics) and
Dr M. A. Hernández (Head of Department of Radiation
Oncology) for their administrative and clinical support with
the equipment. Editorial assistance was provided by
Dr Peter R. Turner, t-SciMed, Reus, Spain.
Conflict of Interest
The study was supported in part by a grant (FUNCIS 98–31)
from the Health and Research Foundation of the Autonomous
Government of the Canary Islands, Spain.
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Received February 29, 2004; accepted August 20, 2004