Inflammation ( # 2015) DOI: 10.1007/s10753-015-0133-1
The Neuroprotective Effect of Glycyrrhizic Acid on an Experimental Model of Focal Cerebral Ischemia in Rats Tarık Akman,1,6 Mustafa Guven,1 Adem Bozkurt Aras,1 Adile Ozkan,2 Halil Murat Sen,2 Ali Okuyucu,3 Yildiray Kalkan,4 Ibrahim Sehitoglu,4 Coskun Silan,5 and Murat Cosar1
Abstract—Cerebral ischemia is still one of the most important topics in neurosciences. Our study aimed to investigate the neuroprotective and anti-oxidant effects of glycyrrhizic acid on focal cerebral ischemia in rats. Twenty-four rats were divided equally into three groups. A middle cerebral artery occlusion model was performed in this study where sham and glycyrrhizic acid were administered intraperitoneally following middle cerebral artery occlusion. Group I was evaluated as control. Malondialdehyde (MDA), superoxide dismutase (SOD), and nuclear respiratory factor-1 (NRF1) levels were analyzed biochemically on the right cerebral hemisphere, while ischemic histopathological studies were completed to investigate the anti-oxidant status. Biochemical results showed that SOD and NRF1 levels were significantly increased in the glycyrrhizic acid group compared with the sham group while MDA levels were significantly decreased. On histopathological examination, cerebral edema, vacuolization, degeneration, and destruction of neurons were decreased in the glycyrrhizic acid group compared with the sham group. Cerebral ischemia was attenuated by glycyrrhizic acid administration. These observations indicate that glycyrrhizic acid may have potential as a therapeutic agent in cerebral ischemia by preventing oxidative stress. KEY WORDS: brain ischemia; glycyrrhizic acid; middle cerebral artery; caspase-3; apoptosis.
INTRODUCTION Brain ischemia is still a serious clinical problem. It may cause permanent neurological disorders and complications. Treatment of brain ischemia is an important topic in neurology. The edema and compression caused by ischemic damage may cause permanent death of brain cells. Understanding the physiopathology of cerebral ischemia is important in terms of taking protective precautions. As 1
Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 2 Department of Neurology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 3 Department of Medical Biochemistry, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey 4 Department of Histology and Embryology, Faculty of Medicine, Recep Tayyip Erdogan University, Rize, Turkey 5 Department of Pharmacology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 6 To whom correspondence should be addressed at Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey. E-mail: [email protected]
brain tissue has high metabolism and rapid glutamic acid cycle, it is very sensitive to ischemic damage. In order, disruption of protein synthesis, brain edema, mitochondrial damage, oxidative stress, increase in free radicals, and cell necrosis and apoptosis play a role in permanent, irreversible brain tissue damage [1]. Polyphenolic compounds are described as chemopreventive agents [2]. In previous years, the discovery of the biological activity of these compounds has increased research into flavonoids and their importance [3]. Glycyrrhizic acid (GA) is a flavonoid group compound isolated from the roots of the Glycyrrhiza glabra (licorice) plant. It has been reported that major bioactive components of licorice are saponins, triterpenes, and flavonoids such as GA and glycyrrhetinic acids [4]. GA is a triterpenesaponin glycoside. Licorice has been used as medicine or food for thousands of years. In fact, licorice root, known as Bsweet root,^ contains the GA compound that is 50 times sweeter than sucrose [5]. GA salt is widely used in sweets, drugs, beverages, chewing gums, chewing tobacco, and toothpastes as flavoring and sweetener [6]. It
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Akman, Guven, Aras, Ozkan, Sen, Okuyucu, Kalkan, Sehitoglu, Silan, and Cosar has been shown that high doses of GA can induce hypertension [7]. However, GA possesses numerous pharmacological effects like anti-inflammatory [8, 9], neuroprotective [10], anti-viral [2], anti-allergic [11], anti-tumor [12], anti-oxidant [13], and hepatoprotective [14] activities. In our study, the anti-oxidant and neuroprotective effects of GA on focal cerebral ischemia in rats were researched. With this aim, the effects of GA on MDA and NRF1 levels and SOD activity after focal cerebral ischemia were studied. Additionally, the dead neurons and the immunopositive cells were counted in the histopathological samples. Toluidine blue staining was used to evaluate general histological examination; caspase-3 and caspase-9 primary antibodies were used to label and evaluate these proteins immunohistochemically.
MATERIALS AND METHODS Animals The rats were supplied from Canakkale Onsekiz Mart University Experimental Research Center. Male Sprague–Dawley rats weighing 300 ± 25 g from 8 to 12 weeks old were used in the experiment. Rats were kept for a week before the experiment to adapt to the environment. This study was conducted in Canakkale Onsekiz Mart University Experimental Research Center. A standard pellet diet (Bil-Yem Ltd., Ankara, Turkey) and tap water were provided ad libitum. Rats were provided with a photoperiodic medium with temperature (23 ± 2 °C), humidity (60 ± 5 %), and white fluorescent tube lighting (hours 8:00 a.m.–8:00 p.m. bright, 8:00 p.m.–8:00 a.m. dark). Experiments were carried out with the permission of Canakkale Onsekiz Mart University Animal Ethics Committee in accordance with the BGuide for the Care and Use of Laboratory Animals (8th edition, 2011)^ (number 2013/03–04). Rats were randomly divided into three equal groups (consisting of eight rats each): Group 1 Control group (n=8, no medication or surgical procedure) Group 2 Sham group (n=8, focal cerebral ischemia was performed via a middle cerebral artery occlusion (MCAO) model. Single-dose, 1 ml, 20 % ethanol was administered intraperitoneally at the fifth minute. Rats were sacrificed at the 24th hour) Group 3 GA group (n=8, intraperitoneal 80 mg/kg GA administered following MCAO at the fifth minute. Rats were sacrificed at the 24th hour)
Before the rats were sacrificed, a craniotomy was performed and the complete brain was obtained. At the end of the experiment, all animals were deeply anesthetized with ketamine (50–60 mg/kg) and sacrificed. Brain tissue was investigated histopathologically, immunohistochemically, and biochemically.
Surgical Procedure A Biopac MP36 (BIOPAC Systems, Inc. Goleta, CA, USA) device was used as a monitor. Mean arterial pulse was 375 per minute during surgery. Body temperature was monitored with a rectal probe and was adjusted to 37.1 to 37.4 °C with a heating pad during surgery. All groups were anesthetized with intramuscular xylazine (5 mg/kg) (Bayer, Istanbul, Turkey) and ketamine hydrochloride (50 mg/kg) (Parke Davis, Istanbul, Turkey) at room temperature with spontaneous respiration. Anesthesia was continued with ketamine injections at intervals without intubation or mechanical ventilation. The rat’s neck was incised under sterile conditions on the operating table in supine position with a right paramedian skin and subdermis incision. Focal cerebral ischemia via the MCAO model was induced with intraluminal filament technique as previously described by Hata et al. [15]. To induce MCAO, 4/0 nylon monofilament suture material (Ethilon Inc., Somerville, NJ, USA) was threaded intraluminally through a small incision in the carotid artery bifurcation until 18–22 mm distal to the right internal carotid artery. Five minutes after MCAO, group 3 rats were given intraperitoneal 80 mg/kg GA. Animals were fed a standard diet and water ad libitum in their cages after surgery. At the end of the 24th hour, all rats were anesthetized with intramuscular ketamine (50 mg/kg). Immediately before sacrifice, the brains of the rats were obtained by craniotomy. As soon as the brain was obtained, the intraluminal monofilament suture advancing to the anterior cerebral artery and occluding the MCA was observed. The brain was washed with ice-cold 0.9 % saline and then dried with filter paper. Half of the right hemisphere was stored in sterile plastic tubes at −80 °C for tissue biochemistry investigations. The other half of the right hemisphere was fixated in 4 % paraformaldehyde solution for histopathological and immunohistochemical investigation. Dosage The dosage was determined as 80 mg/kg body weight based on preliminary studies with various doses
Effect of Glycyrrhizic Acid on a Model of Focal Cerebral Ischemia (10, 25, 50, and 70 mg) to reveal the biological effects of GA [4, 16]. Reagents GA (Catalog No. 50531) was obtained from SigmaAldrich’s Turkey distributor (Interlab, Istanbul, Turkey). GA was dispersed with 20 % ethanol. Modified Lowry Protein Assay Kit (Catalog No. 23240) was obtained from Thermo Scientific Inc. (Waltham, MA, USA). SOD Assay Kit (Catalog No. 706002) was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Rat Malondialdehyde ELISA Kit (Cat. No. CK-E30266) and Rat Nuclear Respiratory Factor 1 ELISA Kit (Cat. No. CKE90555) were obtained from Hangzhou Eastbiopharm Co. Ltd. (Hangzhou, China). Anti-caspase-3 antibody (ab4051) and anti-caspase-9 antibody (ab32539) were obtained from Abcam plc (Cambridge, UK). Evaluation of Neurological Status Neurological status of animals was assessed blindly by a neurologist at the 1st, 12th, and 24th hour after ischemia. To assess the motor function of rats after ischemia, rats were scored by Bederson [17] as follows: scale 0, no deficit; 1, mild forelimb weakness; 2, severe forelimb weakness, consistently turns to side of deficit when lifted by tail; 3, compulsory circling; 4, unconscious; and 5, dead. Tissue Biochemical Examination Tissue samples were pulverized with liquid nitrogen and then homogenized with 1 ml cold PBS on ice at 20,000 rpm for 30 s. The homogenate was centrifuged at 10,000 rpm and 4 °C for 10 min. The supernatant was removed to another tube. All samples were stored at −80 °C until examined. On the day of study, the samples were defrosted to 2–8 °C. Estimation of Levels of Protein, SOD, MDA, and NRF1 Levels The protein content was measured by the method of Lowry et al. with bovine serum albumin as the standard. Results are presented as milligrams per milliliter [18]. Tissue SOD activity was measured with a modified spectrophotometric method at 560 nm as described by Sun et al. [19]. SOD activity is reported as U/ml/mg protein. MDA levels were analyzed for lipid peroxidation products, and results are expressed as nmol/ml/mg tissue.
Tissue MDA levels were determined according to Buege’s ELISA method [20]. NRF1 activates the expression of some key genes regulating cell growth and nuclear genes necessary for both mitochondrial DNA transcription and replication, not only heme biosynthesis but also respiration. NRF1 controls the apoptosis, proliferation, migration, and cellular distribution and adhesion of target genes. NRF1 levels were measured with the ELISA method [21, 22]. Results are reported as ng/ml/mg protein. Histopathologic Examination The brains were fixed with 10 % neutral formaldehyde solution. After 24 h in fixative, they were washed for 6–8 h in running water and rinsed with ethanol-xylene series for automatic tissue tracking (Citadel 2000, Thermo Fisher Scientific Shandon, England) and they were submerged in liquid paraffin. To stain the tissues with hematoxylin and eosin and luxol fast blue, 4–6-μm-thickness sections were made. For immunohistochemical staining, 3–4-μm-thickness sections were made. In the evaluation of hematoxylin and eosin- and luxol fast blue-stained sections, the dead neurons which were characterized by karyolitic, karyorectic nucleus, and vacuolated cytoplasm were counted in 15 different random areas under a ×20 objective magnification. Each area was 1200 μm2 with remaining neuronal cells in the area counted to determine cell density. All the sections were evaluated under a light microscope (Eclipse E-600 Nikon, Japan) and photographed. Sections made for immunohistochemical staining were left in xylene for 20 min, after rinsing with alcohol (70–99 %), then left in 3 % H2O2 solution for 10 min. After washing with PBS, they were heated four times at 700–800 W for 5–10 min in citrate buffer solution and Table 1. The Activity of Superoxide Dismutase and Level of Malondialdehyde and Nuclear Respiratory Factor-1 in Control, Sham, and GA Groups in Ischemic Brain Tissue Groups
SOD (U/ml)
MDA (nmol/ml)
NRF1 (ng/ml)
Control Sham GA p value
6.90 ± 1.20 2.25 ± 0.51a 5.45 ± 1.10a,b
The Neuroprotective Effect of Glycyrrhizic Acid on an Experimental Model of Focal Cerebral Ischemia in Rats Tarık Akman,1,6 Mustafa Guven,1 Adem Bozkurt Aras,1 Adile Ozkan,2 Halil Murat Sen,2 Ali Okuyucu,3 Yildiray Kalkan,4 Ibrahim Sehitoglu,4 Coskun Silan,5 and Murat Cosar1
Abstract—Cerebral ischemia is still one of the most important topics in neurosciences. Our study aimed to investigate the neuroprotective and anti-oxidant effects of glycyrrhizic acid on focal cerebral ischemia in rats. Twenty-four rats were divided equally into three groups. A middle cerebral artery occlusion model was performed in this study where sham and glycyrrhizic acid were administered intraperitoneally following middle cerebral artery occlusion. Group I was evaluated as control. Malondialdehyde (MDA), superoxide dismutase (SOD), and nuclear respiratory factor-1 (NRF1) levels were analyzed biochemically on the right cerebral hemisphere, while ischemic histopathological studies were completed to investigate the anti-oxidant status. Biochemical results showed that SOD and NRF1 levels were significantly increased in the glycyrrhizic acid group compared with the sham group while MDA levels were significantly decreased. On histopathological examination, cerebral edema, vacuolization, degeneration, and destruction of neurons were decreased in the glycyrrhizic acid group compared with the sham group. Cerebral ischemia was attenuated by glycyrrhizic acid administration. These observations indicate that glycyrrhizic acid may have potential as a therapeutic agent in cerebral ischemia by preventing oxidative stress. KEY WORDS: brain ischemia; glycyrrhizic acid; middle cerebral artery; caspase-3; apoptosis.
INTRODUCTION Brain ischemia is still a serious clinical problem. It may cause permanent neurological disorders and complications. Treatment of brain ischemia is an important topic in neurology. The edema and compression caused by ischemic damage may cause permanent death of brain cells. Understanding the physiopathology of cerebral ischemia is important in terms of taking protective precautions. As 1
Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 2 Department of Neurology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 3 Department of Medical Biochemistry, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey 4 Department of Histology and Embryology, Faculty of Medicine, Recep Tayyip Erdogan University, Rize, Turkey 5 Department of Pharmacology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 6 To whom correspondence should be addressed at Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey. E-mail: [email protected]
brain tissue has high metabolism and rapid glutamic acid cycle, it is very sensitive to ischemic damage. In order, disruption of protein synthesis, brain edema, mitochondrial damage, oxidative stress, increase in free radicals, and cell necrosis and apoptosis play a role in permanent, irreversible brain tissue damage [1]. Polyphenolic compounds are described as chemopreventive agents [2]. In previous years, the discovery of the biological activity of these compounds has increased research into flavonoids and their importance [3]. Glycyrrhizic acid (GA) is a flavonoid group compound isolated from the roots of the Glycyrrhiza glabra (licorice) plant. It has been reported that major bioactive components of licorice are saponins, triterpenes, and flavonoids such as GA and glycyrrhetinic acids [4]. GA is a triterpenesaponin glycoside. Licorice has been used as medicine or food for thousands of years. In fact, licorice root, known as Bsweet root,^ contains the GA compound that is 50 times sweeter than sucrose [5]. GA salt is widely used in sweets, drugs, beverages, chewing gums, chewing tobacco, and toothpastes as flavoring and sweetener [6]. It
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Akman, Guven, Aras, Ozkan, Sen, Okuyucu, Kalkan, Sehitoglu, Silan, and Cosar has been shown that high doses of GA can induce hypertension [7]. However, GA possesses numerous pharmacological effects like anti-inflammatory [8, 9], neuroprotective [10], anti-viral [2], anti-allergic [11], anti-tumor [12], anti-oxidant [13], and hepatoprotective [14] activities. In our study, the anti-oxidant and neuroprotective effects of GA on focal cerebral ischemia in rats were researched. With this aim, the effects of GA on MDA and NRF1 levels and SOD activity after focal cerebral ischemia were studied. Additionally, the dead neurons and the immunopositive cells were counted in the histopathological samples. Toluidine blue staining was used to evaluate general histological examination; caspase-3 and caspase-9 primary antibodies were used to label and evaluate these proteins immunohistochemically.
MATERIALS AND METHODS Animals The rats were supplied from Canakkale Onsekiz Mart University Experimental Research Center. Male Sprague–Dawley rats weighing 300 ± 25 g from 8 to 12 weeks old were used in the experiment. Rats were kept for a week before the experiment to adapt to the environment. This study was conducted in Canakkale Onsekiz Mart University Experimental Research Center. A standard pellet diet (Bil-Yem Ltd., Ankara, Turkey) and tap water were provided ad libitum. Rats were provided with a photoperiodic medium with temperature (23 ± 2 °C), humidity (60 ± 5 %), and white fluorescent tube lighting (hours 8:00 a.m.–8:00 p.m. bright, 8:00 p.m.–8:00 a.m. dark). Experiments were carried out with the permission of Canakkale Onsekiz Mart University Animal Ethics Committee in accordance with the BGuide for the Care and Use of Laboratory Animals (8th edition, 2011)^ (number 2013/03–04). Rats were randomly divided into three equal groups (consisting of eight rats each): Group 1 Control group (n=8, no medication or surgical procedure) Group 2 Sham group (n=8, focal cerebral ischemia was performed via a middle cerebral artery occlusion (MCAO) model. Single-dose, 1 ml, 20 % ethanol was administered intraperitoneally at the fifth minute. Rats were sacrificed at the 24th hour) Group 3 GA group (n=8, intraperitoneal 80 mg/kg GA administered following MCAO at the fifth minute. Rats were sacrificed at the 24th hour)
Before the rats were sacrificed, a craniotomy was performed and the complete brain was obtained. At the end of the experiment, all animals were deeply anesthetized with ketamine (50–60 mg/kg) and sacrificed. Brain tissue was investigated histopathologically, immunohistochemically, and biochemically.
Surgical Procedure A Biopac MP36 (BIOPAC Systems, Inc. Goleta, CA, USA) device was used as a monitor. Mean arterial pulse was 375 per minute during surgery. Body temperature was monitored with a rectal probe and was adjusted to 37.1 to 37.4 °C with a heating pad during surgery. All groups were anesthetized with intramuscular xylazine (5 mg/kg) (Bayer, Istanbul, Turkey) and ketamine hydrochloride (50 mg/kg) (Parke Davis, Istanbul, Turkey) at room temperature with spontaneous respiration. Anesthesia was continued with ketamine injections at intervals without intubation or mechanical ventilation. The rat’s neck was incised under sterile conditions on the operating table in supine position with a right paramedian skin and subdermis incision. Focal cerebral ischemia via the MCAO model was induced with intraluminal filament technique as previously described by Hata et al. [15]. To induce MCAO, 4/0 nylon monofilament suture material (Ethilon Inc., Somerville, NJ, USA) was threaded intraluminally through a small incision in the carotid artery bifurcation until 18–22 mm distal to the right internal carotid artery. Five minutes after MCAO, group 3 rats were given intraperitoneal 80 mg/kg GA. Animals were fed a standard diet and water ad libitum in their cages after surgery. At the end of the 24th hour, all rats were anesthetized with intramuscular ketamine (50 mg/kg). Immediately before sacrifice, the brains of the rats were obtained by craniotomy. As soon as the brain was obtained, the intraluminal monofilament suture advancing to the anterior cerebral artery and occluding the MCA was observed. The brain was washed with ice-cold 0.9 % saline and then dried with filter paper. Half of the right hemisphere was stored in sterile plastic tubes at −80 °C for tissue biochemistry investigations. The other half of the right hemisphere was fixated in 4 % paraformaldehyde solution for histopathological and immunohistochemical investigation. Dosage The dosage was determined as 80 mg/kg body weight based on preliminary studies with various doses
Effect of Glycyrrhizic Acid on a Model of Focal Cerebral Ischemia (10, 25, 50, and 70 mg) to reveal the biological effects of GA [4, 16]. Reagents GA (Catalog No. 50531) was obtained from SigmaAldrich’s Turkey distributor (Interlab, Istanbul, Turkey). GA was dispersed with 20 % ethanol. Modified Lowry Protein Assay Kit (Catalog No. 23240) was obtained from Thermo Scientific Inc. (Waltham, MA, USA). SOD Assay Kit (Catalog No. 706002) was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Rat Malondialdehyde ELISA Kit (Cat. No. CK-E30266) and Rat Nuclear Respiratory Factor 1 ELISA Kit (Cat. No. CKE90555) were obtained from Hangzhou Eastbiopharm Co. Ltd. (Hangzhou, China). Anti-caspase-3 antibody (ab4051) and anti-caspase-9 antibody (ab32539) were obtained from Abcam plc (Cambridge, UK). Evaluation of Neurological Status Neurological status of animals was assessed blindly by a neurologist at the 1st, 12th, and 24th hour after ischemia. To assess the motor function of rats after ischemia, rats were scored by Bederson [17] as follows: scale 0, no deficit; 1, mild forelimb weakness; 2, severe forelimb weakness, consistently turns to side of deficit when lifted by tail; 3, compulsory circling; 4, unconscious; and 5, dead. Tissue Biochemical Examination Tissue samples were pulverized with liquid nitrogen and then homogenized with 1 ml cold PBS on ice at 20,000 rpm for 30 s. The homogenate was centrifuged at 10,000 rpm and 4 °C for 10 min. The supernatant was removed to another tube. All samples were stored at −80 °C until examined. On the day of study, the samples were defrosted to 2–8 °C. Estimation of Levels of Protein, SOD, MDA, and NRF1 Levels The protein content was measured by the method of Lowry et al. with bovine serum albumin as the standard. Results are presented as milligrams per milliliter [18]. Tissue SOD activity was measured with a modified spectrophotometric method at 560 nm as described by Sun et al. [19]. SOD activity is reported as U/ml/mg protein. MDA levels were analyzed for lipid peroxidation products, and results are expressed as nmol/ml/mg tissue.
Tissue MDA levels were determined according to Buege’s ELISA method [20]. NRF1 activates the expression of some key genes regulating cell growth and nuclear genes necessary for both mitochondrial DNA transcription and replication, not only heme biosynthesis but also respiration. NRF1 controls the apoptosis, proliferation, migration, and cellular distribution and adhesion of target genes. NRF1 levels were measured with the ELISA method [21, 22]. Results are reported as ng/ml/mg protein. Histopathologic Examination The brains were fixed with 10 % neutral formaldehyde solution. After 24 h in fixative, they were washed for 6–8 h in running water and rinsed with ethanol-xylene series for automatic tissue tracking (Citadel 2000, Thermo Fisher Scientific Shandon, England) and they were submerged in liquid paraffin. To stain the tissues with hematoxylin and eosin and luxol fast blue, 4–6-μm-thickness sections were made. For immunohistochemical staining, 3–4-μm-thickness sections were made. In the evaluation of hematoxylin and eosin- and luxol fast blue-stained sections, the dead neurons which were characterized by karyolitic, karyorectic nucleus, and vacuolated cytoplasm were counted in 15 different random areas under a ×20 objective magnification. Each area was 1200 μm2 with remaining neuronal cells in the area counted to determine cell density. All the sections were evaluated under a light microscope (Eclipse E-600 Nikon, Japan) and photographed. Sections made for immunohistochemical staining were left in xylene for 20 min, after rinsing with alcohol (70–99 %), then left in 3 % H2O2 solution for 10 min. After washing with PBS, they were heated four times at 700–800 W for 5–10 min in citrate buffer solution and Table 1. The Activity of Superoxide Dismutase and Level of Malondialdehyde and Nuclear Respiratory Factor-1 in Control, Sham, and GA Groups in Ischemic Brain Tissue Groups
SOD (U/ml)
MDA (nmol/ml)
NRF1 (ng/ml)
Control Sham GA p value
6.90 ± 1.20 2.25 ± 0.51a 5.45 ± 1.10a,b
