Accepted Manuscript Hyperbaric oxygen treatment in the experimental spinal cord injury model Onur Yaman, Banu Yaman, Figen Aydın, Ahmet Var, Cüneyt Temiz PII:
S1529-9430(14)00162-4
DOI:
10.1016/j.spinee.2014.02.013
Reference:
SPINEE 55773
To appear in:
The Spine Journal
Received Date: 4 January 2012 Revised Date:
3 January 2014
Accepted Date: 3 February 2014
Please cite this article as: Yaman O, Yaman B, Aydın F, Var A, Temiz C, Hyperbaric oxygen treatment in the experimental spinal cord injury model, The Spine Journal (2014), doi: 10.1016/j.spinee.2014.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Hyperbaric oxygen treatment in the experimental spinal cord injury model
1
RI PT
Onur Yaman1, Banu Yaman2, Figen Aydın3, Ahmet Var4, Cüneyt Temiz5
Specialist in Neurosurgery, Tepecik Educational and Training Hospital,İzmir,
2
Specialist in Pathology, Aegean University, Medical Faculty, Department of
3
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Pathology, İzmir, TURKEY
4
SC
TURKEY
Specialist in Hyperbaric medicine, Neoks Co., İzmir, TURKEY
Professor in Clinical Biochemistry, Celal Bayar University, Medical Faculty, Department of Biochemistry, Manisa, TURKEY
Professor in Neurosurgery, Celal Bayar University, Medical Faculty, Department of
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Neurosurgery, Manisa, TURKEY
Onur Yaman
Specialist in Neurosurgery, Tepecik Educational Training Hospital, İzmir, TURKEY
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Correspondence:
E-mail: [email protected] Tel: 090 232 4696969 Fax: 090 232 4696969 Mobile: 090 506 5998527
Acknowledgements
ACCEPTED MANUSCRIPT The authors thank Wanda Reese (of Jade Medical Communications Group, Los Angeles, CA USA) who provided professional English-language editing of this article prior to its final
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acceptance for publication
ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Abstract
2
Background Context: Spinal cord trauma is a major cause of mortality and morbidity.
3
While no known treatment for spinal cord injury (SCI) exists, a limited number of
4
effective treatment modalities and procedures are available that improve secondary
5
injury. Hyperbaric oxygen treatment (HBO) has been used to assist in neurological
6
recovery following cranial injury or ischemic stroke.
7
Purpose: To report findings on the effectiveness of HBO treatment on rats with
8
experimental traumatic spinal cord injury (TSCI). Improvement was evaluated through
9
motor strength assessment and nitrite levels assay testing.
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1
Study Design: We randomly distributed 40 rats among five groups of eight rats each:
11
sham, incurable trauma, induced trauma; HBO treatment begun at the first hour, HBO
12
treatment begun at the sixth hour, and HBO treatment begun at the 24th hour.
13
Method: The HBO treatment was administered to rats in three of the groups, and
14
conducted in two 90-minute sessions, under an absolute atmospheric pressure (ATA) of
15
2.4 at 100% oxygen for five days. In the motor strength evaluations, all of the rats were
16
observed during the inclined plane test, and clinical motor examination on the first, third
17
and fifth days. In addition, the nitrite levels of spinal cord tissues on the sixth days were
18
also studied (No conflict of interest).
19
Results: Results from the inclined plane levels and motor strength test from all three
20
groups undergoing HBO treatment were higher than those from Group 2. It was also
21
determined that early HBO treatment resulted in higher recovery rates (Groups 3 and 4).
22
The highest levels were seen in the group in which the HBO treatments were started in
23
the first hour (Group 3). It was noted that nitrite levels of rats in the group exposed to
24
trauma increased, compared to the sham group; but increased levels also diminished
25
following HBO treatments. Again, the greatest decrease in nitrite levels was evident in
26
the group where the HBO treatment was started the earliest (Group 3).
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Conclusion: Prompt HBO treatment following trauma, significantly contributed to the
2
clinical, histopathological and biochemical recovery of the rats.
3
Key Words: Hyperbaric oxygen treatment, spinal cord injury, rat, nitrite.
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Introduction:
2
The spinal cord is unable to regenerate itself. Permanent damage occurs in patients who
3
have experienced spinal cord injuries. (1, 2).
4
There are two mechanisms that increase the risk of further damage in spinal cord
5
injuries (3, 4, 5, and 6):
6
1-Primary, mechanical injury
7
2-Secondary, ischemic injury
8
Mechanical harm occurs at the time of the event. These injuries cause damage to the
9
nerves, the spinal cord itself and/or spinal vascular structures (7). Secondary injury is
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1
the harm sustained by metabolic and biochemical factors occurring within hours
11
following the primary injury (8, 9). Ischemia is one of the most significant factors
12
leading to secondary injury with the chief problem in the early periods being inadequate
13
perfusion (5, 10). Energy failure following the ischemia leads to a decrease in ATP
14
levels, followed by the commencement of anaerobic respiration. Fehlings and Tator
15
state that ischemia following trauma constitutes the basis of secondary injury, but
16
ischemia is a treatable and revocable process (1). The primary purpose of all
17
experimental and clinical studies on TSCI is to reduce secondary injury.
18
Although the total number of pathological mechanisms caused by spinal cord injuries is
19
not precisely known, processes such as nitric oxide (NO) accumulation, resulting from
20
increases in both calcium, and free radicals are mentioned (7, 12, 13, 14, 15, 16, 17).
21
Lipid peroxidation is considered to be the principal cause among them (18). These
22
oxidative processes begin as a result of increases in hydrogen peroxide, superoxide ions
23
and NO, which cause oxidative damage to lipids, nucleic acid and proteins, with the
24
destructive potential of these free radicals increasing further as they raise endogenous
25
antioxidants in the body (19). Of these free radicals, NO is a primary molecule which
26
plays a key role in many physiological processes such as vascular tonus regulation,
27
thrombocyte functions, neuronal communication and body defense (20). NO and its
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
metabolites are reported to increase, especially in inflammatory and infectious
2
conditions. It is understood that excessive NO production in cerebral ischemia and
3
epilepsy leads to neurotoxicity (21). NO is primarily an unstable gas, which rapidly
4
turns to nitrite, nitrate and peroxynitrite compounds (21, 22). Because its half-life of
5
metabolization is so short, tissue levels are difficult to determine. Levels of stable nitrite
6
and nitrate end products may indirectly affect decisions regarding NO tissue levels. This
7
method was used, for years, to determine NO tissue levels especially in experimental
8
ischemia and reperfusion studies (21).
9
HBO is one possible treatment and support approach following, or prior to, secondary
SC
RI PT
1
injury, and is administered in a closed pressure chamber, under pressure higher than one
11
atmosphere allowing the patient to breathe pure oxygen through an oxygen mask,
12
respiration cap, oxygen tent or endotracheal tube. This is used to contribute to
13
neurological recovery following brain injury and cerebral ischemia (21, 23, 24, 25, 26).
14
The first experimental study in which HBO treatment was administered to spinal
15
traumas was reported by Maeda (27). HBO use for therapeutic purposes was seen for
16
the first time in the study by Hartzog (28).
17
Within the scope of our study, trauma and sham groups were set up to determine the
18
rats’ motor strength and nitrite levels in tissues, following experimental rat spinal cord
19
trauma. To reduce spinal cord ischemia, HBO treatment was administered at the first
20
hour, sixth hour and 24th hour following trauma. Differences among the groups were
21
compared by measurements taken from nitrite levels, motor strength evaluations, and
22
histopathological examinations of tissues, based on a tissue destruction scale.
23
Materials and Methods:
24
Surgical operations within the scope of the study were performed in research
25
laboratories on experimental animals at the Aegean University Hospital and Celal Bayar
26
University Hospital, with permission given by the 9 Eylul University Ethics Committee
27
of Experimental Animals, reference number 23/2009. In total, 40 Sprague-Dawley rats
28
(8 rats in each of the 5 groups) were used for the study. In each group, the rats weighed
29
between 200 to 250 grams, and were grouped randomly to form 5 groups:
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Group 1: The (Sham) group - laminectomy only. No trauma or hyperbaric oxygen
2
treatment administered.
3
Group 2: The (Trauma) group - laminectomy, and SCI.
4
Group 3: Laminectomy, SCI, and hyperbaric oxygen treatment administered in the first
5
hour following trauma.
6
Group 4: Laminectomy, SCI, and hyperbaric oxygen treatment administered in the sixth
7
hour following trauma.
8
Group 5: Laminectomy and SCI, with hyperbaric oxygen treatment administered in the
9
24th hour following trauma.
10
Groups are shown in table 1.
11
Anesthesia:
12
All rats in the groups that underwent surgeries were injected with 2 mgr/ kg ketamine
13
HCI (Ketalar, Parke-Davis Eczacıbaşı-İstanbul) + xylazine intraperitoneally for general
14
anesthesia.
15
Surgery:
16
Intramuscular Sefotaxim was prophylactically given (Bilim Pharmaceuticals- Istanbul)
17
40 mg /kg, 30 minutes before the operation. Rats were positioned prone on a fixing
18
table. Thoracic areas were first sterilized with PVD (Batticon solution, Adeka-Samsun)
19
and shaved. The skins were again sterilized with PVD iodine after shaving. Throughout
20
the operations and until the anesthetic effects were complete, body temperatures were
21
kept at 37 °C with a heating pad. Basing of the interscapular distance incision was
22
acquired through the skin and subcutaneous tissues, via a 2 cm incision at the level of
23
T7-T11. Muscles of paravertebrae were scraped bilaterally. Lamina was observed at this
24
level, and total laminectomy was performed at T9. Through the Tator method, spinal
25
damage was executed in four groups (Figures 1, 2, 3). For purposes of standard trauma,
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
dura and spinal cords were thoroughly clipped via Yasargil aneurysm clip (50±2 grams
2
closing pressure).
3
Following bleeding control, skin tissues were primarily sutured according to anatomic
4
layers. To determine bladder, urination and motor functions, all the rats were put into
5
separate cages and fed standard food stuffs at an appropriate room temperature. Group 2
6
rats, that underwent laminectomies, and in which spinal cord injuries were caused, were
7
not treated. However, eight rats in group 3 underwent HBO treatment at the first hour,
8
eight rats in group 4 at the sixth hour, and eight rats in group 5 at the 24th hour,
9
respectively. Group 1 underwent laminectomy with no spinal cord trauma.
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HBO treatment:
11
Experimental hyperbaric chambers were used for HBO treatment of Group 3, Group 4
12
and Group 5. Rats were treated in these chambers twice a day for five days. Treatment
13
began in Group 3 at the first hour, in Group 4 at the sixth hour and in Group 5 at the
14
24th hour following trauma (the study design is shown in Figure 9). Prior to treatment,
15
chambers were washed for 5 minutes with 100% oxygen, and the desired treatment
16
depth was achieved after 5 minutes (Diving phase). 100% oxygen, at a pressure of 2.4
17
ATA, was administered for 80 minutes (Treatment phase). In the final stage, pressure
18
was decreased (Output phase). Each protocol was completed in 90 minutes. Protocol
19
was determined by hyperbaric medical specialists with expertise in experimental
20
compartment syndrome, and diabetic foot ulcers (29, 30).
21
Humane Termination of Laboratory Animals:
22
The subjects were killed on the sixth day via intraperitoneal thiopental sodium
23
(Pentothal-Abbott Pharmaceuticals-Istanbul) administration.
24
Evaluation of the functional recovery:
25
Inclined plane test:
26
Functional recovery of the rats was evaluated by the inclined plane method which is
27
commonly used in experimental SCI studies (5). In this technique, the rats were put on a
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
tray parallel to a flat surface. The unstable tray edge was raised so that the incline level
2
is increased. The highest angle during which the rats could stay stable for 5 seconds was
3
the inclined plane angle. Rats were motivated by food stuffs given during the test.
4
Within the scope of this study, inclined plane tests were applied to all rats on the first,
5
third and fifth days following surgeries.
6
Clinical motor examination:
7
All rats underwent regular motor examinations to evaluate their functional recovery.
8
The motor functions of rats following surgery were evaluated in compliance with
9
Drummond and Moore criteria (Table 2) on the first, third and fifth days.
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Nitrite (NO2-) Levels Assay:
11
At the end of the sixth day, the rats were positioned supine and anesthetized via 2
12
mgr/kg ketamine and xylazine solution injections intraperitoneally. Following
13
anesthesia, previous incisions were opened, and 5 mm spinal cord segments at the T8-9
14
levels, were resected for purposes of nitrite assay. In the sham group, tissue samples
15
were resected in the middle of the laminectomy sites. Tissues were centrifuged, and
16
their supernatants archived. The Diazotization method was applied in order to determine
17
assay nitrite levels. This method was applied based on the fact that acid medium nitrite
18
and sulphonamide form a pink complex that reacts with N-(1-naphytl) ethylene diamine
19
and whose absorption was measured via a 50 nm wavelength spectrophotometer. The
20
response, known as the Griess reaction, is clearly defined and specific to nitrite. Nitrite
21
and nitrate, oxidation products of NO, are mixed and so nitrate reductase is added to the
22
reaction environment, and nitrate turns into nitrite. The supernatants were poured into
23
3ml propylene tubes, each tube containing 18 microliters of supernatants, and 132
24
microliters of distilled water; 60 microliters of phosphate buffer, 30 microliters of
25
NADPH solution, 30 microliters of FAD solution, and 30 microliters of nitrate
26
reductase were poured over the supernatants. The mixture was incubated in the dark at
27
room temperature. Fresh Griess reagent of 600 microliters was poured over it, and the
28
mixture was incubated for 10 minutes more at room temperature. The absorbance of the
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pink color was measured at 540 nm via spectrophotometer. Each sample was measured
2
twice, and the average was applied.
3
Histolopathologic examination:
4
At the end of the sixth day, all rats anesthetized with intraperitoneally injected ketamine
5
and xylazine solutions described previously, and secured in a prone position on the
6
operating table. Skin sutures were opened, muscles of paravertebrae were divided, and 5
7
millimeters long spinal cord segments were separated. All specimens were put into 10%
8
phosphate buffered formalin solutions for fixation. Subsequently, formalin fixed-
9
paraffin embedded tissue samples were cut into 40-100 nanometers thick sections.
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1
Tissue sections were stained with hematoxylin-eosin and evaluated by light microscopy
11
under 40 and 100 times magnification (Olympus CH 40-Japan) by two pathologists.
12
According to observations, a semiquantitative grading scale was conducted in table 3.
13
The Semiquantitave Grading Method is an often used method in pathological studies to
14
simplify the examination process and to clearly show statistical differences. The
15
histological sections were evaluated by two pathologists in this study. The total score
16
was calculated between 0-12 points. From 0-3 points was labeled grade 1 which
17
indicated minimal bleeding and necrosis. Between 4-7 points was labeled grade 2
18
indicating moderate tissue damage. Grade 3 is between 8-12 points, referring to heavy
19
tissue damage.
20
Statistical Analysis:
21
To evaluate the findings of our study; the SPSS (Statistical Package for Social Sciences)
22
for Windows 13.0 was used. During the data evaluation of the study, the Kruskal-Wallis
23
test was applied to the intergroup comparison of parameters, as the parameters did not
24
comply with a normal distribution during the quantitative data comparison. The
25
Student t Test was applied to detect the group causing dissimilarity. Wilcoxon Signed-
26
Rank Test was applied to the intragroup comparison of parameters. Results were
27
evaluated at 95% confidence level and the significance level was p≤ 0.05.
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Results:
2
Following surgery, it was observed that 32 rats, in which spinal cord trauma had been
3
caused, became paraplegic. Further observation noted that the motor strength of 8 rats in
4
the sham group, which went through laminectomy only, were healthy.
5
Evaluation of the Inclined Plane Test:
6
The inclined plane levels of groups on the first, third and fifth days are shown in table 4.
7
When the groups’ values were compared, a statistically significant difference was
8
observed between the sham group and the trauma group, as well as the treatment groups
9
(Student t Test, p
S1529-9430(14)00162-4
DOI:
10.1016/j.spinee.2014.02.013
Reference:
SPINEE 55773
To appear in:
The Spine Journal
Received Date: 4 January 2012 Revised Date:
3 January 2014
Accepted Date: 3 February 2014
Please cite this article as: Yaman O, Yaman B, Aydın F, Var A, Temiz C, Hyperbaric oxygen treatment in the experimental spinal cord injury model, The Spine Journal (2014), doi: 10.1016/j.spinee.2014.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Hyperbaric oxygen treatment in the experimental spinal cord injury model
1
RI PT
Onur Yaman1, Banu Yaman2, Figen Aydın3, Ahmet Var4, Cüneyt Temiz5
Specialist in Neurosurgery, Tepecik Educational and Training Hospital,İzmir,
2
Specialist in Pathology, Aegean University, Medical Faculty, Department of
3
M AN U
Pathology, İzmir, TURKEY
4
SC
TURKEY
Specialist in Hyperbaric medicine, Neoks Co., İzmir, TURKEY
Professor in Clinical Biochemistry, Celal Bayar University, Medical Faculty, Department of Biochemistry, Manisa, TURKEY
Professor in Neurosurgery, Celal Bayar University, Medical Faculty, Department of
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5
Neurosurgery, Manisa, TURKEY
Onur Yaman
Specialist in Neurosurgery, Tepecik Educational Training Hospital, İzmir, TURKEY
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1
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Correspondence:
E-mail: [email protected] Tel: 090 232 4696969 Fax: 090 232 4696969 Mobile: 090 506 5998527
Acknowledgements
ACCEPTED MANUSCRIPT The authors thank Wanda Reese (of Jade Medical Communications Group, Los Angeles, CA USA) who provided professional English-language editing of this article prior to its final
AC C
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acceptance for publication
ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Abstract
2
Background Context: Spinal cord trauma is a major cause of mortality and morbidity.
3
While no known treatment for spinal cord injury (SCI) exists, a limited number of
4
effective treatment modalities and procedures are available that improve secondary
5
injury. Hyperbaric oxygen treatment (HBO) has been used to assist in neurological
6
recovery following cranial injury or ischemic stroke.
7
Purpose: To report findings on the effectiveness of HBO treatment on rats with
8
experimental traumatic spinal cord injury (TSCI). Improvement was evaluated through
9
motor strength assessment and nitrite levels assay testing.
M AN U
SC
RI PT
1
Study Design: We randomly distributed 40 rats among five groups of eight rats each:
11
sham, incurable trauma, induced trauma; HBO treatment begun at the first hour, HBO
12
treatment begun at the sixth hour, and HBO treatment begun at the 24th hour.
13
Method: The HBO treatment was administered to rats in three of the groups, and
14
conducted in two 90-minute sessions, under an absolute atmospheric pressure (ATA) of
15
2.4 at 100% oxygen for five days. In the motor strength evaluations, all of the rats were
16
observed during the inclined plane test, and clinical motor examination on the first, third
17
and fifth days. In addition, the nitrite levels of spinal cord tissues on the sixth days were
18
also studied (No conflict of interest).
19
Results: Results from the inclined plane levels and motor strength test from all three
20
groups undergoing HBO treatment were higher than those from Group 2. It was also
21
determined that early HBO treatment resulted in higher recovery rates (Groups 3 and 4).
22
The highest levels were seen in the group in which the HBO treatments were started in
23
the first hour (Group 3). It was noted that nitrite levels of rats in the group exposed to
24
trauma increased, compared to the sham group; but increased levels also diminished
25
following HBO treatments. Again, the greatest decrease in nitrite levels was evident in
26
the group where the HBO treatment was started the earliest (Group 3).
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Conclusion: Prompt HBO treatment following trauma, significantly contributed to the
2
clinical, histopathological and biochemical recovery of the rats.
3
Key Words: Hyperbaric oxygen treatment, spinal cord injury, rat, nitrite.
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1
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Introduction:
2
The spinal cord is unable to regenerate itself. Permanent damage occurs in patients who
3
have experienced spinal cord injuries. (1, 2).
4
There are two mechanisms that increase the risk of further damage in spinal cord
5
injuries (3, 4, 5, and 6):
6
1-Primary, mechanical injury
7
2-Secondary, ischemic injury
8
Mechanical harm occurs at the time of the event. These injuries cause damage to the
9
nerves, the spinal cord itself and/or spinal vascular structures (7). Secondary injury is
M AN U
SC
RI PT
1
the harm sustained by metabolic and biochemical factors occurring within hours
11
following the primary injury (8, 9). Ischemia is one of the most significant factors
12
leading to secondary injury with the chief problem in the early periods being inadequate
13
perfusion (5, 10). Energy failure following the ischemia leads to a decrease in ATP
14
levels, followed by the commencement of anaerobic respiration. Fehlings and Tator
15
state that ischemia following trauma constitutes the basis of secondary injury, but
16
ischemia is a treatable and revocable process (1). The primary purpose of all
17
experimental and clinical studies on TSCI is to reduce secondary injury.
18
Although the total number of pathological mechanisms caused by spinal cord injuries is
19
not precisely known, processes such as nitric oxide (NO) accumulation, resulting from
20
increases in both calcium, and free radicals are mentioned (7, 12, 13, 14, 15, 16, 17).
21
Lipid peroxidation is considered to be the principal cause among them (18). These
22
oxidative processes begin as a result of increases in hydrogen peroxide, superoxide ions
23
and NO, which cause oxidative damage to lipids, nucleic acid and proteins, with the
24
destructive potential of these free radicals increasing further as they raise endogenous
25
antioxidants in the body (19). Of these free radicals, NO is a primary molecule which
26
plays a key role in many physiological processes such as vascular tonus regulation,
27
thrombocyte functions, neuronal communication and body defense (20). NO and its
AC C
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10
3
ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
metabolites are reported to increase, especially in inflammatory and infectious
2
conditions. It is understood that excessive NO production in cerebral ischemia and
3
epilepsy leads to neurotoxicity (21). NO is primarily an unstable gas, which rapidly
4
turns to nitrite, nitrate and peroxynitrite compounds (21, 22). Because its half-life of
5
metabolization is so short, tissue levels are difficult to determine. Levels of stable nitrite
6
and nitrate end products may indirectly affect decisions regarding NO tissue levels. This
7
method was used, for years, to determine NO tissue levels especially in experimental
8
ischemia and reperfusion studies (21).
9
HBO is one possible treatment and support approach following, or prior to, secondary
SC
RI PT
1
injury, and is administered in a closed pressure chamber, under pressure higher than one
11
atmosphere allowing the patient to breathe pure oxygen through an oxygen mask,
12
respiration cap, oxygen tent or endotracheal tube. This is used to contribute to
13
neurological recovery following brain injury and cerebral ischemia (21, 23, 24, 25, 26).
14
The first experimental study in which HBO treatment was administered to spinal
15
traumas was reported by Maeda (27). HBO use for therapeutic purposes was seen for
16
the first time in the study by Hartzog (28).
17
Within the scope of our study, trauma and sham groups were set up to determine the
18
rats’ motor strength and nitrite levels in tissues, following experimental rat spinal cord
19
trauma. To reduce spinal cord ischemia, HBO treatment was administered at the first
20
hour, sixth hour and 24th hour following trauma. Differences among the groups were
21
compared by measurements taken from nitrite levels, motor strength evaluations, and
22
histopathological examinations of tissues, based on a tissue destruction scale.
23
Materials and Methods:
24
Surgical operations within the scope of the study were performed in research
25
laboratories on experimental animals at the Aegean University Hospital and Celal Bayar
26
University Hospital, with permission given by the 9 Eylul University Ethics Committee
27
of Experimental Animals, reference number 23/2009. In total, 40 Sprague-Dawley rats
28
(8 rats in each of the 5 groups) were used for the study. In each group, the rats weighed
29
between 200 to 250 grams, and were grouped randomly to form 5 groups:
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ACCEPTED MANUSCRIPT Hyperbaric oxygen treatment in the experimental spinal cord injury model
Group 1: The (Sham) group - laminectomy only. No trauma or hyperbaric oxygen
2
treatment administered.
3
Group 2: The (Trauma) group - laminectomy, and SCI.
4
Group 3: Laminectomy, SCI, and hyperbaric oxygen treatment administered in the first
5
hour following trauma.
6
Group 4: Laminectomy, SCI, and hyperbaric oxygen treatment administered in the sixth
7
hour following trauma.
8
Group 5: Laminectomy and SCI, with hyperbaric oxygen treatment administered in the
9
24th hour following trauma.
10
Groups are shown in table 1.
11
Anesthesia:
12
All rats in the groups that underwent surgeries were injected with 2 mgr/ kg ketamine
13
HCI (Ketalar, Parke-Davis Eczacıbaşı-İstanbul) + xylazine intraperitoneally for general
14
anesthesia.
15
Surgery:
16
Intramuscular Sefotaxim was prophylactically given (Bilim Pharmaceuticals- Istanbul)
17
40 mg /kg, 30 minutes before the operation. Rats were positioned prone on a fixing
18
table. Thoracic areas were first sterilized with PVD (Batticon solution, Adeka-Samsun)
19
and shaved. The skins were again sterilized with PVD iodine after shaving. Throughout
20
the operations and until the anesthetic effects were complete, body temperatures were
21
kept at 37 °C with a heating pad. Basing of the interscapular distance incision was
22
acquired through the skin and subcutaneous tissues, via a 2 cm incision at the level of
23
T7-T11. Muscles of paravertebrae were scraped bilaterally. Lamina was observed at this
24
level, and total laminectomy was performed at T9. Through the Tator method, spinal
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damage was executed in four groups (Figures 1, 2, 3). For purposes of standard trauma,
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dura and spinal cords were thoroughly clipped via Yasargil aneurysm clip (50±2 grams
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closing pressure).
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Following bleeding control, skin tissues were primarily sutured according to anatomic
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layers. To determine bladder, urination and motor functions, all the rats were put into
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separate cages and fed standard food stuffs at an appropriate room temperature. Group 2
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rats, that underwent laminectomies, and in which spinal cord injuries were caused, were
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not treated. However, eight rats in group 3 underwent HBO treatment at the first hour,
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eight rats in group 4 at the sixth hour, and eight rats in group 5 at the 24th hour,
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respectively. Group 1 underwent laminectomy with no spinal cord trauma.
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HBO treatment:
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Experimental hyperbaric chambers were used for HBO treatment of Group 3, Group 4
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and Group 5. Rats were treated in these chambers twice a day for five days. Treatment
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began in Group 3 at the first hour, in Group 4 at the sixth hour and in Group 5 at the
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24th hour following trauma (the study design is shown in Figure 9). Prior to treatment,
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chambers were washed for 5 minutes with 100% oxygen, and the desired treatment
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depth was achieved after 5 minutes (Diving phase). 100% oxygen, at a pressure of 2.4
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ATA, was administered for 80 minutes (Treatment phase). In the final stage, pressure
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was decreased (Output phase). Each protocol was completed in 90 minutes. Protocol
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was determined by hyperbaric medical specialists with expertise in experimental
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compartment syndrome, and diabetic foot ulcers (29, 30).
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Humane Termination of Laboratory Animals:
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The subjects were killed on the sixth day via intraperitoneal thiopental sodium
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(Pentothal-Abbott Pharmaceuticals-Istanbul) administration.
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Evaluation of the functional recovery:
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Inclined plane test:
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Functional recovery of the rats was evaluated by the inclined plane method which is
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commonly used in experimental SCI studies (5). In this technique, the rats were put on a
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tray parallel to a flat surface. The unstable tray edge was raised so that the incline level
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is increased. The highest angle during which the rats could stay stable for 5 seconds was
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the inclined plane angle. Rats were motivated by food stuffs given during the test.
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Within the scope of this study, inclined plane tests were applied to all rats on the first,
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third and fifth days following surgeries.
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Clinical motor examination:
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All rats underwent regular motor examinations to evaluate their functional recovery.
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The motor functions of rats following surgery were evaluated in compliance with
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Drummond and Moore criteria (Table 2) on the first, third and fifth days.
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Nitrite (NO2-) Levels Assay:
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At the end of the sixth day, the rats were positioned supine and anesthetized via 2
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mgr/kg ketamine and xylazine solution injections intraperitoneally. Following
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anesthesia, previous incisions were opened, and 5 mm spinal cord segments at the T8-9
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levels, were resected for purposes of nitrite assay. In the sham group, tissue samples
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were resected in the middle of the laminectomy sites. Tissues were centrifuged, and
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their supernatants archived. The Diazotization method was applied in order to determine
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assay nitrite levels. This method was applied based on the fact that acid medium nitrite
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and sulphonamide form a pink complex that reacts with N-(1-naphytl) ethylene diamine
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and whose absorption was measured via a 50 nm wavelength spectrophotometer. The
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response, known as the Griess reaction, is clearly defined and specific to nitrite. Nitrite
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and nitrate, oxidation products of NO, are mixed and so nitrate reductase is added to the
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reaction environment, and nitrate turns into nitrite. The supernatants were poured into
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3ml propylene tubes, each tube containing 18 microliters of supernatants, and 132
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microliters of distilled water; 60 microliters of phosphate buffer, 30 microliters of
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NADPH solution, 30 microliters of FAD solution, and 30 microliters of nitrate
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reductase were poured over the supernatants. The mixture was incubated in the dark at
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room temperature. Fresh Griess reagent of 600 microliters was poured over it, and the
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mixture was incubated for 10 minutes more at room temperature. The absorbance of the
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pink color was measured at 540 nm via spectrophotometer. Each sample was measured
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twice, and the average was applied.
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Histolopathologic examination:
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At the end of the sixth day, all rats anesthetized with intraperitoneally injected ketamine
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and xylazine solutions described previously, and secured in a prone position on the
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operating table. Skin sutures were opened, muscles of paravertebrae were divided, and 5
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millimeters long spinal cord segments were separated. All specimens were put into 10%
8
phosphate buffered formalin solutions for fixation. Subsequently, formalin fixed-
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paraffin embedded tissue samples were cut into 40-100 nanometers thick sections.
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Tissue sections were stained with hematoxylin-eosin and evaluated by light microscopy
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under 40 and 100 times magnification (Olympus CH 40-Japan) by two pathologists.
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According to observations, a semiquantitative grading scale was conducted in table 3.
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The Semiquantitave Grading Method is an often used method in pathological studies to
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simplify the examination process and to clearly show statistical differences. The
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histological sections were evaluated by two pathologists in this study. The total score
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was calculated between 0-12 points. From 0-3 points was labeled grade 1 which
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indicated minimal bleeding and necrosis. Between 4-7 points was labeled grade 2
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indicating moderate tissue damage. Grade 3 is between 8-12 points, referring to heavy
19
tissue damage.
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Statistical Analysis:
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To evaluate the findings of our study; the SPSS (Statistical Package for Social Sciences)
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for Windows 13.0 was used. During the data evaluation of the study, the Kruskal-Wallis
23
test was applied to the intergroup comparison of parameters, as the parameters did not
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comply with a normal distribution during the quantitative data comparison. The
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Student t Test was applied to detect the group causing dissimilarity. Wilcoxon Signed-
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Rank Test was applied to the intragroup comparison of parameters. Results were
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evaluated at 95% confidence level and the significance level was p≤ 0.05.
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Results:
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Following surgery, it was observed that 32 rats, in which spinal cord trauma had been
3
caused, became paraplegic. Further observation noted that the motor strength of 8 rats in
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the sham group, which went through laminectomy only, were healthy.
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Evaluation of the Inclined Plane Test:
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The inclined plane levels of groups on the first, third and fifth days are shown in table 4.
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When the groups’ values were compared, a statistically significant difference was
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observed between the sham group and the trauma group, as well as the treatment groups
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(Student t Test, p
