Original Paper Pharmacology 2014;94:41–50 DOI: 10.1159/000365219
Received: April 28, 2014 Accepted after revision: June 12, 2014 Published online: August 28, 2014
Effects of Intravenous Human Umbilical Cord Blood Mesenchymal Stem Cell Therapy versus Gabapentin in Pentylenetetrazole-Induced Chronic Epilepsy in Rats Amira S. Mohammed Mohammed M. Ewais Mona K. Tawfik Soha S. Essawy Department of Pharmacology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
Abstract Aim: The identification and application of stem cells to treat central nervous system disorders represent a dramatic evolution and expansion into the realms of neurorestoration and neuroregeneration. The aim of this study was to assess the possible ameliorative effect of mesenchymal stem cells (MSCs) in comparison to gabapentin on pentylenetetrazole (PTZ)-induced epileptogenesis and its consequences. Methods: Thirty-two rats were divided into 4 equal groups; group I: saline-injected group, group II: PTZ group, which received 13 intraperitoneal (i.p.) injections of PTZ (30 mg/kg) 3 times/ week, groups III and IV: groups received PTZ and were treated with i.p. gabapentin (200 mg/kg) 60 min before each PTZ injection (group III) or a single intravenous injection of 106 MSCs/rat at day 22 (group IV). Results: Treatment with either gabapentin or MSCs demonstrated a significant improvement in the PTZ-induced epileptogenesis and its severe consequences, i.e. oxidative stress damage, motor and cognitive
© 2014 S. Karger AG, Basel 0031–7012/14/0942–0041$39.50/0 E-Mail [email protected] www.karger.com/pha
impairments. Moreover, they enhanced the GABA neurotransmitter levels. Meanwhile, MSC administration to chronic epileptic rats afforded more ameliorative effects on PTZ-induced epileptogenesis and its severe consequences in comparison to gabapentin. Conclusion: These data indicate that MSCs were superior to gabapentin in ameliorating PTZ-induced epileptogenesis and verified the potential use of MSCs in seizure control, motor and cognitive impairments, oxidative stress, and the impairing GABA level in experimentally induced epilepsy. © 2014 S. Karger AG, Basel
Introduction
Epilepsy is the second most common neurologic disorder after stroke [1]. Although standard therapy permits control of seizures in 80% of all patients, millions have uncontrolled epilepsy [2]. In patients refractive to treatment, brain surgery is the most important alternative treatment; however, surgery includes risks and costs that have to be considered. Pharmacotherapy has a high success rate in terms of seizure relief, but it comes with the Soha S. Essawy Department of Pharmacology, Faculty of Medicine Suez Canal University Ismailia 41522 (Egypt) E-Mail sohaessawy @ yahoo.com
Downloaded by: University of Tokyo 157.82.153.40 - 5/16/2015 8:26:54 PM
Key Words Epilepsy · Gabapentin · Mesenchymal stem cells · Pentylenetetrazole · Rats
Methods Experimental Animals Thirty-two adult female Wistar rats weighing 170 ± 30 g were used in this study. They were purchased from the Egyptian Organization for Biological Products and Vaccines. The rats were housed in clean well-ventilated stainless steel cages, main-
42
Pharmacology 2014;94:41–50 DOI: 10.1159/000365219
tained on a normal light-dark cycle and at a temperature of 25 ± 3 ° C throughout the experiment and left 1 week for acclimatization. Standard rodent chow and water ad libitum were provided. The protocol for stem cell collection, separation and DNA extraction was reviewed and approved by the local Ethics Committee. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Suez Canal University (Ismailia, Egypt), following the National Institutes of Health guide for the care and use of laboratory animals (USA).
Drugs and Chemicals PTZ (Sigma-Aldrich Chemical Co., Egypt) and gabapentin (Delta Pharm Co., Egypt) were dissolved in normal saline. The chemicals needed for MSCs separation and purification were purchased from Lonza Bioproducts Co. (Belgium). PTZ-Induced Kindling The rats received a total of 13 subconvulsive intraperitoneal (i.p.) injections of PTZ (30 mg/kg) 3 times/week [11]. Experimental Design The animals were randomly allocated into 4 groups of 8 rats each. Group I: saline control group; the rats received i.p. saline injections parallel to the PTZ injections at a final volume of 2 ml/kg. Group II: epileptic group; the rats were subjected to a total of 13 i.p. injections of PTZ (30 mg/kg) 3 times/week [11]. Group III: gabapentin-treated group; the kindled rats received gabapentin (200 mg/kg i.p.) 60 min before each PTZ injection [12]. Group IV: MSC-treated group; the kindled rats received a single intravenous (i.v.) dose of human umbilical cord blood (HUCB)derived MSCs (1 × 106 cells/rat) in the tail vein, beginning after 22 days of PTZ injections (just after the 10th injection) [13]. Technique of Collection, Separation, and Transplantation of Mononuclear Cells Collection of HUCB After obtaining informed consent, HUCB was collected ex utero from healthy full-term pregnant women with male fetuses during normal delivery. HUCB was collected by 4 volunteers in four 50-ml graded sterile plastic Falcon tubes (Greiner Bio-One, Germany) containing 7 ml citrate phosphate dextrose adenine-1 anticoagulant. Immediately after cutting the cord, and before placental separation, the maternal end of the umbilical cord was sterilized using ethyl alcohol and the blood was allowed to flow into the tubes. The blood samples were kept at a temperature of 4 ° C during the transport and storage procedures, until they were processed within 24 h after collection [14].
Separation and Purification of Mononuclear Cells from HUCB Step I: Isolation of Low-Density Mononuclear Cells. HUCB was kept in tubes with anticoagulant and was diluted 1:1 in isolation buffer [100 ml phosphate-buffered saline + 10 ml RPM1 + 1 ml 10% fetal calf serum (all Lonza Bioproducts Co.)]. Seven milliliter diluted HUCB was layered drop by drop using a sterile Pasteur pipette into a sterile centrifuge tube (50 ml; Greiner Bio-One) containing 15 ml lymphocyte separation medium, FicollHypaque solution (Sigma-Aldrich Chemical Co., catalog No.
Mohammed/Ewais/Tawfik/Essawy
Downloaded by: University of Tokyo 157.82.153.40 - 5/16/2015 8:26:54 PM
cost of a frequent reliance on toxicity and memory deficits as reported in many studies [3]. Unlike pharmacotherapy, cell transplantation therapy has the potential for anti-epileptogenic or diseasemodifying effects by directly targeting seizure foci or propagation pathways [4]. Additionally, it can replace the loss of GABA neurotransmitter and glial cells associated with chronic epilepsy [5]. Stem cell-based therapy for neurodegenerative diseases has been widely investigated as has cell replacement; both aim at replacing the degenerated neurons, providing a better environment for damaged tissue, and saving the remaining neurons [6]. Stem cell transplantation benefits are now attributed to an attenuation of inflammation, a neuroprotection of the damaged milieu through growth factor and chemokine secretion to promote cell survival and proliferation, and an enhancement of endogenous recovery processes [7]. In chronic epilepsy, human pluripotent cell-derived neural stem cells have been reported to migrate into the injured dentate gyrus and differentiate into neurons, glia, and predifferentiated GABA-ergic cells [3, 5]. The kindling phenomenon is considered an animal model of epileptogenesis. Kindling can be induced by repeated administration of convulsant chemicals such as pentylenetetrazole (PTZ) [8]. PTZ interacts competitively with the picrotoxin-binding site of the GABAA receptor, thereby decreasing the chloride flux across the membrane and inducing generalized tonic-clonic seizure [9]. Gabapentin – an analogue of GABA – has an antiepileptic effect by altering the GABA metabolism, its nonsynaptic release, or its reuptake by GABA transporters [10]. So, the aim of this study was to investigate the possible ameliorative role of mesenchymal stem cells (MSCs) in comparison to gabapentin on PTZ-induced epileptogenesis and its severe consequences. As neurorestoration and replacing neurons or glial cell types destroyed by damage or chronic disease are an extremely active area of investigation, our study was extended to evaluate the effect of MSCs and gabapentin on the GABA neurotransmitter level and the expression of the glial cell marker S100β protein.
Functional Assessment All animals were observed for 30 min, after each PTZ injection, to determine the seizure severity score. Additionally, motor coordination and cognitive function were assessed. The Seizure Severity Score The seizure severity score was assessed in rats using Racine’s scale [20]. This scale describes 6 stages: stage 0: normal nonepileptic activity; stage 1: snout and facial movements, hyperactivity, grooming, sniffing, scratching, and wet dog shakes; stage 2: head nodding, staring, and tremor; stage 3: forelimb clonus and forelimb extension; stage 4: rearing and salivating, and stage 5: falling and status epilepticus. Assessment of Motor Coordination Open Field Test. The rats were put individually in an open field arena (113 × 113 × 44 cm) for 5 min and observed for ambulation frequency and number of stops. In addition, the activity factor A (ambulation frequency/total number of stops) was calculated [21]. Rotarod Test. The rotarod test was run for up to 5 min. The rats were placed on a rotating drum with a diameter of 6 cm (speed 20 rpm). The latency to fall represents a measure for motor coordination [22].
the dark box was punished by an electric foot shock (0.7 mA for 2 s). Twenty-four hours later, the animals were again put into the illuminated box and observed up to 3 min for time elapsing before entering the dark box [23]. Processing of the Brain At the end of the experiment, all rats were anesthetized by a thiopental sodium injection (50 mg/kg) [24] and decapited. The brains were washed in ice-cold saline, and the hippocampus was prepared as 300-μm slices using Vibratome 1000 Plus for the detection of S100β protein [25]. A part of the hippocampus of each rat was immediately frozen at –80 ° C to detect the male-specific SRY gene, oxidant and antioxidant markers as well as the GABA level.
Real-Time PCR Analysis The SRY gene (on the Y chromosome) was used to identify male donor-derived cells in female recipients. Genomic DNA was extracted by Jena Bioscience (Germany). Real-time PCR was done using Light Cycler (Biocompare, Germany). The primer sequences used were: 5′-CAGCTAACACTGATCTTTTC-3′ and 5′-TTACTGAGCCAGAATCATAG-3′ (Light Cycler-DNA Amplification Kit SYBR Green I; Biocompare). DNA from normal human females and males was used as negative and positive controls, respectively [26]. Determination of Oxidant and Antioxidant Markers in the Brains 100 mg of each brain was homogenized. The homogenate was centrifuged at 2,000 g for 10 min. The supernatant was used for the analysis of malondialdehyde (MDA) [27], reduced glutathione (GSH) [28], superoxide dismutase (SOD), and catalase (CATA) [29, 30] using a UV-visible spectrophotometer (UV-1601PC; Shimadzu, Japan). Determination of GABA Level by HPLC Frozen tissue was homogenized for 20 s in 500 μl ice-cold mobile phase containing 3,4-dihydroxybenzylamine as internal standard. The suspension was centrifuged at 10,000 g for 1 min, and the supernatant was removed and filtered for the determination of the GABA level, using the reverse-phase HPLC coupled to an electrochemical detector as previously described [31]. Immunohistochemistry Assay Detection of S100β Protein S100β is used as a marker for glial cell differentiation. Primary antibodies to S100β were applied and incubated for 60 min at room temperature according to the manufacturer’s protocol. 3,3′-Diaminobenzidine was used as a chromogen substrate. Mayer’s hematoxylin was used as a counter stain. The slides were then examined using a light microscope.
Assessment of Cognitive Function Memory impairment was tested by a passive avoidance task. On day 0 (learning trial), the pretreated rats were placed in an illuminated box connected to a dark box (40 × 40 × 30 cm). Entering
Statistical Analysis All results were collected, expressed as mean ± SD, and analyzed using SPSS Software (SPSS Inc., Chicago, Ill., USA). Oneway analysis of variance followed by Duncan’s post hoc test was used to test the significance of the difference between the quantitative variables. A p value
Received: April 28, 2014 Accepted after revision: June 12, 2014 Published online: August 28, 2014
Effects of Intravenous Human Umbilical Cord Blood Mesenchymal Stem Cell Therapy versus Gabapentin in Pentylenetetrazole-Induced Chronic Epilepsy in Rats Amira S. Mohammed Mohammed M. Ewais Mona K. Tawfik Soha S. Essawy Department of Pharmacology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
Abstract Aim: The identification and application of stem cells to treat central nervous system disorders represent a dramatic evolution and expansion into the realms of neurorestoration and neuroregeneration. The aim of this study was to assess the possible ameliorative effect of mesenchymal stem cells (MSCs) in comparison to gabapentin on pentylenetetrazole (PTZ)-induced epileptogenesis and its consequences. Methods: Thirty-two rats were divided into 4 equal groups; group I: saline-injected group, group II: PTZ group, which received 13 intraperitoneal (i.p.) injections of PTZ (30 mg/kg) 3 times/ week, groups III and IV: groups received PTZ and were treated with i.p. gabapentin (200 mg/kg) 60 min before each PTZ injection (group III) or a single intravenous injection of 106 MSCs/rat at day 22 (group IV). Results: Treatment with either gabapentin or MSCs demonstrated a significant improvement in the PTZ-induced epileptogenesis and its severe consequences, i.e. oxidative stress damage, motor and cognitive
© 2014 S. Karger AG, Basel 0031–7012/14/0942–0041$39.50/0 E-Mail [email protected] www.karger.com/pha
impairments. Moreover, they enhanced the GABA neurotransmitter levels. Meanwhile, MSC administration to chronic epileptic rats afforded more ameliorative effects on PTZ-induced epileptogenesis and its severe consequences in comparison to gabapentin. Conclusion: These data indicate that MSCs were superior to gabapentin in ameliorating PTZ-induced epileptogenesis and verified the potential use of MSCs in seizure control, motor and cognitive impairments, oxidative stress, and the impairing GABA level in experimentally induced epilepsy. © 2014 S. Karger AG, Basel
Introduction
Epilepsy is the second most common neurologic disorder after stroke [1]. Although standard therapy permits control of seizures in 80% of all patients, millions have uncontrolled epilepsy [2]. In patients refractive to treatment, brain surgery is the most important alternative treatment; however, surgery includes risks and costs that have to be considered. Pharmacotherapy has a high success rate in terms of seizure relief, but it comes with the Soha S. Essawy Department of Pharmacology, Faculty of Medicine Suez Canal University Ismailia 41522 (Egypt) E-Mail sohaessawy @ yahoo.com
Downloaded by: University of Tokyo 157.82.153.40 - 5/16/2015 8:26:54 PM
Key Words Epilepsy · Gabapentin · Mesenchymal stem cells · Pentylenetetrazole · Rats
Methods Experimental Animals Thirty-two adult female Wistar rats weighing 170 ± 30 g were used in this study. They were purchased from the Egyptian Organization for Biological Products and Vaccines. The rats were housed in clean well-ventilated stainless steel cages, main-
42
Pharmacology 2014;94:41–50 DOI: 10.1159/000365219
tained on a normal light-dark cycle and at a temperature of 25 ± 3 ° C throughout the experiment and left 1 week for acclimatization. Standard rodent chow and water ad libitum were provided. The protocol for stem cell collection, separation and DNA extraction was reviewed and approved by the local Ethics Committee. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Suez Canal University (Ismailia, Egypt), following the National Institutes of Health guide for the care and use of laboratory animals (USA).
Drugs and Chemicals PTZ (Sigma-Aldrich Chemical Co., Egypt) and gabapentin (Delta Pharm Co., Egypt) were dissolved in normal saline. The chemicals needed for MSCs separation and purification were purchased from Lonza Bioproducts Co. (Belgium). PTZ-Induced Kindling The rats received a total of 13 subconvulsive intraperitoneal (i.p.) injections of PTZ (30 mg/kg) 3 times/week [11]. Experimental Design The animals were randomly allocated into 4 groups of 8 rats each. Group I: saline control group; the rats received i.p. saline injections parallel to the PTZ injections at a final volume of 2 ml/kg. Group II: epileptic group; the rats were subjected to a total of 13 i.p. injections of PTZ (30 mg/kg) 3 times/week [11]. Group III: gabapentin-treated group; the kindled rats received gabapentin (200 mg/kg i.p.) 60 min before each PTZ injection [12]. Group IV: MSC-treated group; the kindled rats received a single intravenous (i.v.) dose of human umbilical cord blood (HUCB)derived MSCs (1 × 106 cells/rat) in the tail vein, beginning after 22 days of PTZ injections (just after the 10th injection) [13]. Technique of Collection, Separation, and Transplantation of Mononuclear Cells Collection of HUCB After obtaining informed consent, HUCB was collected ex utero from healthy full-term pregnant women with male fetuses during normal delivery. HUCB was collected by 4 volunteers in four 50-ml graded sterile plastic Falcon tubes (Greiner Bio-One, Germany) containing 7 ml citrate phosphate dextrose adenine-1 anticoagulant. Immediately after cutting the cord, and before placental separation, the maternal end of the umbilical cord was sterilized using ethyl alcohol and the blood was allowed to flow into the tubes. The blood samples were kept at a temperature of 4 ° C during the transport and storage procedures, until they were processed within 24 h after collection [14].
Separation and Purification of Mononuclear Cells from HUCB Step I: Isolation of Low-Density Mononuclear Cells. HUCB was kept in tubes with anticoagulant and was diluted 1:1 in isolation buffer [100 ml phosphate-buffered saline + 10 ml RPM1 + 1 ml 10% fetal calf serum (all Lonza Bioproducts Co.)]. Seven milliliter diluted HUCB was layered drop by drop using a sterile Pasteur pipette into a sterile centrifuge tube (50 ml; Greiner Bio-One) containing 15 ml lymphocyte separation medium, FicollHypaque solution (Sigma-Aldrich Chemical Co., catalog No.
Mohammed/Ewais/Tawfik/Essawy
Downloaded by: University of Tokyo 157.82.153.40 - 5/16/2015 8:26:54 PM
cost of a frequent reliance on toxicity and memory deficits as reported in many studies [3]. Unlike pharmacotherapy, cell transplantation therapy has the potential for anti-epileptogenic or diseasemodifying effects by directly targeting seizure foci or propagation pathways [4]. Additionally, it can replace the loss of GABA neurotransmitter and glial cells associated with chronic epilepsy [5]. Stem cell-based therapy for neurodegenerative diseases has been widely investigated as has cell replacement; both aim at replacing the degenerated neurons, providing a better environment for damaged tissue, and saving the remaining neurons [6]. Stem cell transplantation benefits are now attributed to an attenuation of inflammation, a neuroprotection of the damaged milieu through growth factor and chemokine secretion to promote cell survival and proliferation, and an enhancement of endogenous recovery processes [7]. In chronic epilepsy, human pluripotent cell-derived neural stem cells have been reported to migrate into the injured dentate gyrus and differentiate into neurons, glia, and predifferentiated GABA-ergic cells [3, 5]. The kindling phenomenon is considered an animal model of epileptogenesis. Kindling can be induced by repeated administration of convulsant chemicals such as pentylenetetrazole (PTZ) [8]. PTZ interacts competitively with the picrotoxin-binding site of the GABAA receptor, thereby decreasing the chloride flux across the membrane and inducing generalized tonic-clonic seizure [9]. Gabapentin – an analogue of GABA – has an antiepileptic effect by altering the GABA metabolism, its nonsynaptic release, or its reuptake by GABA transporters [10]. So, the aim of this study was to investigate the possible ameliorative role of mesenchymal stem cells (MSCs) in comparison to gabapentin on PTZ-induced epileptogenesis and its severe consequences. As neurorestoration and replacing neurons or glial cell types destroyed by damage or chronic disease are an extremely active area of investigation, our study was extended to evaluate the effect of MSCs and gabapentin on the GABA neurotransmitter level and the expression of the glial cell marker S100β protein.
Functional Assessment All animals were observed for 30 min, after each PTZ injection, to determine the seizure severity score. Additionally, motor coordination and cognitive function were assessed. The Seizure Severity Score The seizure severity score was assessed in rats using Racine’s scale [20]. This scale describes 6 stages: stage 0: normal nonepileptic activity; stage 1: snout and facial movements, hyperactivity, grooming, sniffing, scratching, and wet dog shakes; stage 2: head nodding, staring, and tremor; stage 3: forelimb clonus and forelimb extension; stage 4: rearing and salivating, and stage 5: falling and status epilepticus. Assessment of Motor Coordination Open Field Test. The rats were put individually in an open field arena (113 × 113 × 44 cm) for 5 min and observed for ambulation frequency and number of stops. In addition, the activity factor A (ambulation frequency/total number of stops) was calculated [21]. Rotarod Test. The rotarod test was run for up to 5 min. The rats were placed on a rotating drum with a diameter of 6 cm (speed 20 rpm). The latency to fall represents a measure for motor coordination [22].
the dark box was punished by an electric foot shock (0.7 mA for 2 s). Twenty-four hours later, the animals were again put into the illuminated box and observed up to 3 min for time elapsing before entering the dark box [23]. Processing of the Brain At the end of the experiment, all rats were anesthetized by a thiopental sodium injection (50 mg/kg) [24] and decapited. The brains were washed in ice-cold saline, and the hippocampus was prepared as 300-μm slices using Vibratome 1000 Plus for the detection of S100β protein [25]. A part of the hippocampus of each rat was immediately frozen at –80 ° C to detect the male-specific SRY gene, oxidant and antioxidant markers as well as the GABA level.
Real-Time PCR Analysis The SRY gene (on the Y chromosome) was used to identify male donor-derived cells in female recipients. Genomic DNA was extracted by Jena Bioscience (Germany). Real-time PCR was done using Light Cycler (Biocompare, Germany). The primer sequences used were: 5′-CAGCTAACACTGATCTTTTC-3′ and 5′-TTACTGAGCCAGAATCATAG-3′ (Light Cycler-DNA Amplification Kit SYBR Green I; Biocompare). DNA from normal human females and males was used as negative and positive controls, respectively [26]. Determination of Oxidant and Antioxidant Markers in the Brains 100 mg of each brain was homogenized. The homogenate was centrifuged at 2,000 g for 10 min. The supernatant was used for the analysis of malondialdehyde (MDA) [27], reduced glutathione (GSH) [28], superoxide dismutase (SOD), and catalase (CATA) [29, 30] using a UV-visible spectrophotometer (UV-1601PC; Shimadzu, Japan). Determination of GABA Level by HPLC Frozen tissue was homogenized for 20 s in 500 μl ice-cold mobile phase containing 3,4-dihydroxybenzylamine as internal standard. The suspension was centrifuged at 10,000 g for 1 min, and the supernatant was removed and filtered for the determination of the GABA level, using the reverse-phase HPLC coupled to an electrochemical detector as previously described [31]. Immunohistochemistry Assay Detection of S100β Protein S100β is used as a marker for glial cell differentiation. Primary antibodies to S100β were applied and incubated for 60 min at room temperature according to the manufacturer’s protocol. 3,3′-Diaminobenzidine was used as a chromogen substrate. Mayer’s hematoxylin was used as a counter stain. The slides were then examined using a light microscope.
Assessment of Cognitive Function Memory impairment was tested by a passive avoidance task. On day 0 (learning trial), the pretreated rats were placed in an illuminated box connected to a dark box (40 × 40 × 30 cm). Entering
Statistical Analysis All results were collected, expressed as mean ± SD, and analyzed using SPSS Software (SPSS Inc., Chicago, Ill., USA). Oneway analysis of variance followed by Duncan’s post hoc test was used to test the significance of the difference between the quantitative variables. A p value
