Neurological damage from spinal cord injury (SCI) is a result of primary mechanical damage and subsequent secondary damage [1,2,3,4]. Oxidative stress occurs during primary damage due to the cellular release of cytoplasmic components and mitochondrial dysfunction and continues throughout secondary damage due to neuroinflammation. Secondary damage includes tissue necrosis from free oxygen radicals, lipid peroxidation products, immune responses, and a complex series of events involving energy metabolism. Oxygen radicals such as O− and OH− ions are highly active free radicals that are usually eliminated after being converted into O2 and H2O2 via antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase. Oxidative stress creates an imbalance between oxidants and antioxidants at the cellular level. An antioxidant defense system protects cells by neutralizing the harmful effects of oxidants and free radicals and high levels of antioxidants have been found in the spinal cord [5, 6]. If an appropriate antioxidant response is not obtained following SCI, further cellular damage occurs, contributing to a positive feedback cycle resulting in further oxidative stress [5]. Oxidative stress is an ongoing process throughout both primary and secondary damage, and the severity and uncontrolled nature of oxidative stress are related to the magnitude of the damage [1, 4,5,6]. Thus, markers of oxidative stress have been used to monitor SCI and response to treatment [7,8,9], and many studies have focused on resolving inflammation and antioxidant therapies for traumatic brain and SCI [6, 10].
Genistein, a phytoestrogen, is an isoflavonoid found in high concentrations in soybeans [11,12,13]. Genistein has been shown to reduce the risk of cancers such as those of the breast and prostate, which are associated with estrogen [11]. In addition, positive effects on bone mineral development, anti-menopausal effects, and antioxidant and anti-inflammatory effects have been reported [13,14,15,16,17,18,19,20]. Although genistein has been shown to have potent antioxidant and anti-inflammatory effects in several studies of brain injury [16, 17], to our knowledge, its effect on SCI-induced damage remains unknown. In the present study, the antioxidant and anti-inflammatory effects of genistein were investigated in a model of SCI in rats, and its contribution to the recovery of the spinal cord was evaluated biochemically, histopathologically, and functionally through oxidant and antioxidant markers.
The protocols for this study were approved by the local ethics committee of Kobay Deney Experimental Animals Laboratory (No. 280). All protocols followed national guidelines for the care and use of laboratory animals and were compliant with the U.S. Health Research Extension Act (Public Law 99–158, 1985 “Animals in Research”). Reporting follows ARRIVE 2.0 guidelines [21]. Adult female Wistar–Hannover albino rats (each weighing 250–300 g; n = 21) were separated into 3 groups of 7 rats without selection. During the experiments, all rats were kept in a standard postoperative care room (at 20–25 °C with 50%–60% humidity grouped in polycarbonate cages) and allowed standard feed, under 12-h light and dark cycles.
A commercial kit was used to determine the total anti-oxidant status (TAS) and total oxidative status (TOS) in the tissue [22, 23]. Tissue protein was assayed using the Lowry method [24]. Oxidative stress index (OSI) was obtained by dividing TOS values by TAS values [OSI (arbitrary Unit) = (TOS, μmol H2O2 eq/L)/(TAS, μmol Trolox eq/L)].
Serum thiol–disulfide (SS) was measured with an automatic analyzer (Cobas 501; Roche) using a method developed by Erel and Neselioglu [25]. Briefly, disulfide bonds in the sample were converted into functional thiol groups with NaBH4. The total thiol (TT) content in the sample was calculated using a modified Ellman reagent. Serum disulfide amount was determined using the following formula: (serum TT − serum native thiol [NT])/2.
Serum ischemia-modified albumin (IMA) levels were determined according to the Bar–Or method [26]. The results are expressed in absorbance units (AbsU). Serum catalase activity was measured using a method described by Góth [27].
The rats were separated into 3 equal number groups without selection (A: control group, B: weight-drop group, C: genistein group) (
In the control group (n = 7), only a T7-8-9 laminectomy was performed.
In the trauma group (n = 7), after a T7-8-9 laminectomy, trauma was induced with a modified Allen weight-drop apparatus. Using a glass tube with a diameter of 0.5 cm and a length of 10 cm, a 10 g weight was dropped on the intact dura from a height of 10 cm.
In the genistein group (n = 7), a T7-8-9 laminectomy was performed, and spinal trauma was induced as described above. Rats in this group were administered a first dose of genistein (Sigma-Aldrich; 2 mg/kg subcutaneously) at 15 min immediately after trauma and further administration of genistein at 2 mg/kg/day for 7 days.
Timeline for the experiment. In group A (n = 7, control group, only T7-8-9 laminectomy), in group B (n = 7, trauma group, after T7-8-9 laminectomy and trauma was induced with a modified Allen weight-drop method, in group C (n = 7, genistein group, T7-8-9 laminectomy and spinal trauma was induced as described above. It was planned that group C would receive the first dose of genistein at 15 min immediately after trauma and would be given genistein subcutaneously at 2 mg/kg/day for 7 days). The rats were killed on the 7th day and the spinal cord was removed as a whole. No rats were lost, and no infection, or additional problems were observed during the experiment. cat, catalase; Cox-2, cyclooxygenase-2; IMA, ischemia-modified albumin; NT, native thiol; OSI, oxidative stress index; SS, disulfide; TT, total thiol; TAS, total antioxidant status; TOS, total oxidative status.
After deep general anesthesia with 1 mg/kg of ketamine and 1 mg/kg of xylazine the rats were killed on the 7th day by transcardiac perfusion with saline followed by phosphate-buffered 4% paraformaldehyde (in 0.1 mol/L phosphate-buffered saline, pH 7.4), and their spinal cords removed. No rats died unintentionally, and no infection or serious adverse events were observed during the experiment.
Tail blood was taken before anesthesia, at the 12th hour, and on the 1st, 3rd, 5th, and 7th days after the surgical procedure. Catalase, IMA, and thiol–disulfide balance were assayed. After killing the rats on the 7th day, the spinal cord tissue in the T7-8-9 region was removed, and TOS and antioxidant capacity (TAS), and OSI determined in the spinal cord tissue samples.
Basso–Beattie and Bresnahan (BBB) scores were examined in all rats at baseline and the 12th hour, and on the 1st, 3rd, 5th, and 7th days. The results were evaluated statistically [28, 29].
After the rats were killed on the 7th day, their entire spinal cords were removed. Next, 1-cm specimens from the traumatized site of each spinal cord were obtained and put into phosphate-buffered 10% formaldehyde. On gross dissection, sections of spinal cords cut perpendicularly were processed and embedded in paraffin blocks. Then, 4-μm sections were deparaffinized in xylene and rehydrated through a graded series of ethanol before hematoxylin and eosin staining and immunohistochemistry.
Immunohistochemistry was performed using a mouse monoclonal cyclo-oxygenase-2 (Cox-2) antibody (clone D-12: catalog No. sc-166475, Santa Cruz Biotechnology; Research Resource Identifier (RRID): AB_2276666) primary antibody with a Leica Bond-Max auto-stain detection system (catalog No. DS9800, Leica) according to the manufacturer’s instructions.
A pathologist, who was blinded to the groups, examined the immunohistochemically stained sections. Cox-2 immunoreactive (IR) cells were counted per 3 high powered fields (HPFs Olympus BX51 microscope 400×) for each spinal cord.
Data were analyzed using IBM SPSS Statistics for Windows (version 25). Data are expressed as mean ± standard deviation (SD) or median (interquartile range; Q1, Q3). A one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test and a Kruskal–Wallis test followed by a Dunn post hoc test were used to analyze differences between independent variables. A repeated measures ANOVA and Friedman analyses with Bonferroni post hoc correction were used to determine the differences between the dependent variables. Differences with
There was no significant difference in the baseline serum activity between the groups. Although no significant difference (
Time-dependent comparison of oxidative and antioxidative serum markers between groups
Catalase (kU/L) | ||||||||
A | 79.0 ± 18.0 | 74.2 ± 9.2 | 31.5 ± 13.8 | 84.3 ± 6.1 | 61.7 ± 12.0 | 86.0 ± 10.0 | <0.001 | |
B | 48.1 ± 9.9 | 74.9 ± 7.9 | 42.6 ± 9.2 | 48.0 ± 4.6 | 54.1 ± 12.3 | 37.9 ± 21.9 | 0.02 | |
C | 51.4 ± 11.9 | 51.8 ± 30.0 | 76.9 ± 13.9 | 69.4 ± 13.3 | 75.0 ± 9.1 | 75.0 ± 9.1 | 0.002 | |
0.11 | 0.11 | 0.001 (x, z) | 0.15 (x, y, z) | 0.15 | 0.001 (x, z) | |||
Ischemia-modified albumin (AbsU) | ||||||||
A | 0.84 ± 0.05 | 0.82 ± 0.05 | 0.99 ± 0.05 | 0.93 ± 0.04 | 0.91 ± 0.05 | 1.01 ± 0.03 | <0.001 | |
B | 0.87 ± 0.04 | 0.93 ± 0.03 | 0.91 ± 0.03 | 0.78 ± 0.08 | 0.92 ± 0.04 | 0.92 ± 0.03 | 0.001 | |
C | 0.91 ± 0.03 | 0.92 ± 0.04 | 0.85 ± 0.04 | 1.00 ± 0.04 | 0.98 ± 0.05 | 1.07 ± 0.09 | <0.001 | |
0.10 | 0.001 (x, y) | <0.001 (x, y, z) | <0.001 (x, y, z) | 0.10 | 0.004 (x, z) | |||
NT (mmol/L) | ||||||||
A | 183.1 ± 37.8 | 110.9 ± 36.0 | 116.0 ± 16.2 | 71.7 ± 13.3 | 157.3 ± 26.0 | 87.4 ± 20.4 | <0.001 | |
B | 190.8 ± 51.0 | 222.2 ± 27.1 | 96.0 ± 31.4 | 127.8 ± 24.3 | 130.7 ± 29.8 | 166.3 ± 22.5 | <0.001 | |
C | 194.7 ± 38.7 | 99.9 ± 37.6 | 167.3 ± 24.7 | 99.8 ± 32.4 | 108.1 ± 28.6 | 71.2 ± 22.9 | <0.001 | |
0.91 | 0.007 (x, y) | 0.002 (y, z) | 0.006 (x) | 0.02 (y) | 0.001 (x, z) | |||
TT (mmol/L) | ||||||||
A | 250.3 ± 53.0 | 189.4 ± 47.5 | 158.5 ± 18.2 | 142.4 ± 12.5 | 202.5 ± 33.0 | 152.3 ± 21.0 | 0.002 | |
B | 248.1 ± 63.7 | 281.7 ± 29.4 | 182.8 ± 54.5 | 207.4 ± 31.4 | 213.0 ± 42.6 | 238.4 ± 40.4 | 0.02 | |
C | 245.0 ± 55.7 | 170.8 ± 23.0 | 210.4 ± 28.2 | 160.1 ± 40.3 | 187.7 ± 42.9 | 106.0 ± 19.7 | 0.02 | |
0.99 | 0.007 (x, z) | 0.04 (y) | 0.01 (x) | 0.51 | <0.001 (x, y, z) | |||
SS (mmol/L) | ||||||||
A | 33.6 ± 10.2 | 39.2 ± 9.5 | 21.2 ± 6.1 | 35.3 ± 5.2 | 22.6 ± 5.0 | 32.4 ± 8.4 | 0.007 | |
B | 28.6 ± 11.3 | 29.8 ± 8.8 | 43.4 ± 14.0 | 39.8 ± 12.5 | 41.2 ± 17.6 | 36.1 ± 11.7 | 0.33 | |
C | 25.2 ± 10.5 | 35.5 ± 18.4 | 21.6 ± 4.2 | 30.2 ± 8.7 | 39.8 ± 9.6 | 17.4 ± 8.5 | 0.005 | |
0.297 | 0.286 | 0.004 (x, z) | 0.193 | 0.014 (x, y) | 0.004 (x, z) | |||
NT/TT (%) | ||||||||
A | 73.4 ± 5.0 | 57.9 ± 6.7 | 73.3 ± 6.9 | 50.2 ± 7.3 | 77.7 ± 3.0 | 57.4 ± 9.8 | <0.001 | |
B | 77.1 ± 5.6 | 78.9 ± 5.7 | 52.5 ± 7.5 | 62.0 ± 9.8 | 62.1 ± 12.3 | 70.2 ± 5.7 | <0.001 | |
C | 70.0 ± 4.9 | 58.6 ± 18.9 | 79.5 ± 3.1 | 61.6 ± 11.5 | 57.4 ± 6.5 | 66.9 ± 18.5 | <0.001 | |
0.12 | 0.003 (x, z) | 0.001 (x, z) | 0.04 (x, y) | 0.005 (x, y) | 0.049 (x) | |||
SS/TT (%) | ||||||||
A | 13.3 ± 2.5 | 21.0 ± 3.4 | 13.3 ± 3.5 | 24.9 ± 3.7 | 11.2 ± 1.5 | 21.3 ± 4.9 | <0.001 | |
B | 11.5 ± 2.8 | 10.5 ± 2.8 | 23.8 ± 3.7 | 19.0 ± 4.9 | 18.9 ± 6.2 | 14.9 ± 2.9 | <0.001 | |
C | 10.0 ± 2.5 | 20.7 ± 9.5 | 10.3 ± 1.5 | 19.2 ± 5.7 | 21.3 ± 3.2 | 16.6 ± 9.2 | <0.001 | |
0.11 | 0.003 (x, z) | 0.001 (x, z) | 0.04 (x, y) | 0.005 (x, y) | 0.049 (x) | |||
SS/NT (%) | ||||||||
A | 18.4 ± 4.7 | 37.3 ± 9.8 | 18.8 ± 7.0 | 51.7 ± 16.3 | 14.5 ± 2.5 | 39.7 ± 17.6 | <0.001 | |
B | 15.2 ± 5.1 | 13.6 ± 4.6 | 46.9 ± 13.5 | 32.6 ± 13.9 | 33.5 ± 18.5 | 21.6 ± 5.6 | <0.001 | |
C | 12.7 ± 3.7 | 45.2 ± 36.4 | 13.0 ± 2.5 | 34.6 ± 22.1 | 38.2 ± 10.4 | 31.1 ± 28.5 | 0.001 | |
0.12 | 0.003 (x, z) | 0.001 (x, z) | 0.04 (x, y) | 0.005 (x, y) | 0.052 (x) |
Group A, control group; group B, trauma group; group C, genistein group; x, significant difference between groups A and B; y, significant difference between groups A and C; z, significant difference between groups B and C;
AbsU, absorbance units; NT, native thiol; NT/TT, native thiol/total thiol; SS, disulfide; SS/TT, disulfide/total thiol; TT, total thiol.
Catalase serum levels. Significant difference was observed on the 7th day (
No significant difference in serum levels of IMA was observed between the 3 groups at baseline (
There was no significant difference in the baseline serum SS serum levels between the groups. No significant difference (
SS serum levels. Significant difference was observed on the 1st, 5th, and 7th day between the control group (A white bars) and trauma group (B light gray bars) and genistein group (C dark gray bars), and between groups B and C (
There was no significant difference in the baseline serum levels of NT between the groups. The level of NT in group C was significantly lower than those of groups A and B at the 12th hour (
There was no significant difference in the baseline TT serum levels between the groups. A significant difference in TT values was observed between the groups at the 12th hour (
No significant difference in ratios was observed between the groups at baseline. There was a significant difference between the groups at all time points (12th hour,
No significant difference in the ratio was observed between the groups at baseline. There was a significant difference in the ratio between the groups at all time points (12th hour,
These parameters were evaluated in the spinal cord tissue obtained on the 7th day. There was no significant difference between the groups for TAS (
Time-dependent comparison of TAS, TOS, OSI, and Cox-2 immunoreactivity between the groups
TAS (nmol Trolox Eq/mg protein) | 35.8 (30.7–40.8) | 32.7 (28.6–36.8) | 30.8 (29.8–35.3) | 0.26 |
TOS (nmol H2O2 Eq/mg protein) | 1.08 (0.59–1.12) | 0.67 (0.58–0.85) | 0.83 (0.60–1.11) | 0.40 |
OSI (TOS/TAS) | 0.03 ± 0.01 | 0.02 ± 0.01 | 0.03 ± 0.01 | 0.56 |
Cox 2 IR cells/3 HPFs | 19.1 ± 5.3 | 17.1 ± 5.9 | 7.71 ± 5.0 | 0.008 (y, z) |
Data are expressed as mean ± SD or median (Q1, Q3). Cox-2, cyclooxygenase-2; HPF, 400× high-powered field; IR, immunoreactive; OSI, oxidative stress index; TAS, total antioxidant status; TOS, total oxidative status; x, significant difference between groups A and B; y, significant difference between groups A and C; z, significant difference between groups B and C. Repeated measures ANOVA and Friedman analyses for (dependent) variables within the groups are indi cated as follows (
Group A: serum catalase activity on the 1st day was significantly lower than that at baseline, at the 12th hour, and activity on the 3rd and 7th days. Activity on the 5th day was significantly lower than that on the 3rd and 7th days. Group B: the activity at the 12th hour was significantly higher than that at baseline, and on the 1st, 3rd, 5th, and 7th days. Group C: the activity at baseline and at the 12th hour was significantly lower than that on the 1st, 3rd, 5th, and 7th days (
Group A: baseline and 12th hour levels were significantly lower than those on the 1st, 3rd, 5th, and 7th days. Group B: the IMA level on the 3rd day was significantly lower than that on the 12th hour, 1st, 5th, and 7th days. Group C: the level on the 1st day was significantly lower than that on the 3rd, 5th, and 7th days.
Group A: the serum NT level at baseline was significantly higher than that at the 12th hour, and on the 1st, 3rd, and 7th days. The level on the 3rd day was significantly lower than that at the 12th hour, and on the 1st and 5th days. The level on the 5th day was significantly higher than that on the 7th day. Group B: the levels at baseline and at the 12th hour were significantly higher than those on the 1st, 3rd, and 5th days. The level on the 1st day was significantly higher than those on the 3rd, 5th, and 7th days. Group C: the serum NT level on the 7th day was significantly lower than that at baseline and on the 1st day.
Group A: the serum level of TT at baseline was significantly higher than that at the 12th hour, and on the 1st, 3rd, 5th, and 7th days. The level on the 3rd day was significantly lower than that on the 5th day. Group B: the level at the 12th hour was significantly higher than that on the 1st and 3rd days. Group C: the serum level of TT on the 7th day was significantly lower than that at baseline, at the 12th hour, and the levels on the 1st, 3rd, and 5th days.
Group A: the level of serum disulfide at the 12th hour was significantly higher than that on the 1st and 5th days. Group B: no significant temporal differences in serum disulfide levels were found. Group C: the level on the 5th day was significantly higher than that on the 1st and 7th days (
Group A: the ratio at the 12th hour, and those on the 3th and 7th days were significantly lower than those at baseline, and on the 1st and 7th days. Group B: the ratio on the 1st day was significantly lower than that at baseline and at the 12th hour. Group C: the ratios at the 12th hour and on the 5th day were significantly lower than those at baseline and on the 1st day.
Group A: the ratio on the 3rd day was significantly higher than those at baseline, and on the 1st and 5th days. The ratio on the 5th day was significantly lower than that at the 12th hour and on the 7th day. Group B: the ratio on the 1st day was significantly higher than that at baseline, at the 12th hour, and on the 7th day. Group C: the ratio at the 12th hour was significantly higher than that at baseline and on the 1st day.
Group A: the ratio on the 3rd day was significantly higher than that at baseline, and on the 1st and 5th days. The ratio on the 5th day was significantly lower than that at the 12th hour and on the 7th day. Group B: the ratio on the 1st day was significantly higher than that at baseline, at the 12th hour, and on the 7th day. Group C: the ratio at the 12th hour was significantly higher than that at baseline and on the 1st day.
A significant difference was found between the groups (
Immunohistochemically stained rat spinal cord sections with hematoxylin and eosin counterstain. A mouse monoclonal Cox-2 antibody (clone D-12: catalog No. sc-166475, Santa Cruz Biotechnology; RRID: AB_2276666) was used for primary detection, the monoclonal antibody was visualized using a diaminobenzidine chromogen system showing brown staining. Cox-2 positive cells were counted per 3 HPFs for each spinal cord (left panel, trauma group (B), right panel, genistein group (C). Left panel scale bar indicates 0.5 mm, inset scale bar indicates 50 μm. Right panel scale bar indicates 0.5 mm. Cox-2, cyclo-oxygenase-2; HPFs, 400× high-powered fields; RRID, research resource identifier.
Cox-2 immunoreactivity scoring. Significant difference (
At the 12th hour, the trauma (B) and genistein (C) groups had significantly (
Time-dependent comparison of BBB scores in rats
12th hour | 7 (6–7) | 5 (3–6) | 5 (2–6) | 0.006 (x, y) |
1st day | 13 (12–13) | 11 (9–12) | 11 (10–12) | 0.002 (x, y) |
3rd day | 13 (13–13) | 11 (9–12) | 12 (11–13) | 0.001 (x, y, z) |
5th day | 13 (13–13) | 11 (9–12) | 13 (12–13) | <0.001 (x, z) |
7th day | 21 (21–21) | 18 (14–19) | 21 (21–21) | <0.001 (x, z) |
<0.001 | <0.001 | <0.001 |
Data are expressed median (interquartile range; Q1, Q3). x, significant difference between groups A and B; y, significant difference between groups A and C; z, significant difference between groups B and C).
BBB, Basso–Beattie and Bresnahan.
Friedman analyses of BBB scores were as follows. Group A: 12th hour score was significantly lower than that on the 1st, 3rd, 5th, and 7th days. The score on the 7th day was significantly higher than that on the 1st, 3rd, and 5th days. Group B: 12th hour score was significantly lower than that on the 1st, 3rd, 5th, and 7th days. The score on the 7th day was significantly higher than that on the 1st, 3rd, 5th days. Group C: 12th hour score was lower than that on the 1st, 3rd, 5th, and 7th days. The score on the 7th day was significantly higher than that on the 1st, 3rd, 5th days (
In the present study, although there was no difference between the groups at the 12th hour (
IMA has been considered as a marker of oxidative processes. Oxygen radicals resulting from trauma damage the
The thiol component containing sulfhydryl (-SH) is important for the antioxidative impact of oxidative processes. Thiol groups of some sulfur-containing amino acids are oxidized by reactive oxygen radicals and are converted reversibly into disulfide bonds. Levels indicated by NT are thiols that are normally found in plasma. The sum of the NTs and thiols formed by the reduction of disulfides is TT. Therefore, the increase in disulfide values represents an increase in the oxidant capacity, and the increase in thiol values represents an increase in the antioxidant capacity [25, 36,37,38]. Dynamic thiol/disulfide balance has been used frequently in clinical studies because of its antioxidant effect and its effect on apoptosis [5, 36,37,38]. In the present study, compared with the control group, there was a significant difference between the trauma and genistein groups, particularly on the 7th day (
Cox-2 is a conditionally-induced enzyme that is expressed in the event of inflammatory damage [39]. Cox-2 plays a role in the inflammation in secondary damage related to SCI. Cox-2 mRNA and protein expression were increased following SCI, and selective inhibition of Cox-2 led to improved functional outcomes, and neuronal cell death and neuro-inflammation were suppressed in experimental models [40]. In addition, Cox-2 expression decreases with the increase in antioxidant defense and decreases under oxidative stress [41, 42]. Genistein inhibits Cox-2 in laboratory studies [43]. The present study found a significant difference in Cox-2 expression between the genistein group and the control and trauma groups. A lower number of Cox-2 IR cells were found in spinal cords from rats in the genistein group than in those from the trauma and control groups (
Functional assessment of the rats showed that the baseline functional outcomes were worse in the trauma and genistein groups compared with those in the control group, but rats in the genistein group achieved better functional outcomes than those in the trauma group over time. Moreover, on the 5th day and thereafter, the significant difference between the control and trauma groups persisted, whereas a significant difference between genistein and the control groups was not found. This result showed that better functional outcomes were achieved in the genistein group.
The 7-day follow-up period in the present study was short, limiting its prediction of long-term outcomes of trauma. In addition, the induced model of spinal cord trauma is not a model of severe spinal cord trauma, limiting its prediction of severe SCI outcomes. The study is limited by using the BBB score alone to evaluate function. Although oxidative stress markers are considered to be appropriate for assessment in this model in rats, a problem is that they are highly variable in patients with multiple traumas, and may not be useful in clinical practice.
Genistein has antioxidant, protective, and anti-inflammatory effects in an experimental model of SCI in rats. Further study of genistein is warranted as it shows potential as a therapeutic agent for the clinical treatment of SCI.