Saad Habib-E-Rasul Mullah, Jahanara Urmy, Motoki Inaji, Tadashi Nariai, Satoru Ishibashi, Kikuo Ohno
After traumatic brain injury (TBI), increased concentration of extracellular adenosine is considered as one of the role playing causes in the secondary brain damages. We examined if administration of adenosine A2A receptor antagonist may have efficacy in ameliorating neurological symptom by blocking secondary brain damage through cascades initiated by adenosine A2a receptor. Moderate fluid percussion brain injury was induced in Mongolian gerbils. Medication groups received i.p. injection of SCH58261 (selective adenosine A2A receptor antagonist) for different durations. Open-field locomotion test and grabbing test were conducted before and 1, 3, 5, 7 and 9 days after injury. Moderate TBI increased the total distance of movement at all time points. Administration of SCH58261 significantly blocked hyperlocomotion that was observed after TBI. There was no significant difference in total distance moved among the trauma-medication and sham groups. In the grabbing test, grabbing time was significantly increased in TBI group on 3, 5, 7 and 9days after the operation. SCH58261 also improved grabbing time. Adenosine A2A antagonist successfully suppressed the trauma-induced hyperlocomotion presumably by blocking secondary brain damage.
Traumatic brain injury (TBI) causes a spectrum of clinical outcomes ranging from death, disability to behavior abnormalities, learning difficulties, cognitive dysfunction.
Primary impact of TBI is followed by cascade of mechanical and chemical changes leading to secondary damages. In the course of TBI an increase in adenosine is detected both in the intracerebral   and in the cerebrospinal fluid levels  . Intracerebral adenosine rises due to metabolic failure in most types of brain insults such as ischemia, TBI, inflammation, cytotoxicity etc.   . Adenosine neuromodulation in turn, affects the concentration of other neurotransmitters such as Glutamate, GABA, aspartae, taurine and other excitatory amino acids  . Raised level of adenosine after TBI has shown correlation with high mortality .
In ischemic rat, Adenosine A2A receptor antagonism has showed improved outcome, with a reduction in the release of glutamate, GABA and other neurotransmitters . Different adenosine receptor modulations have shown to have improved outcome after TBI. But the specific role of a selective adenosine A2A antagonist has not yet been checked. In our study, we checked the effect A2A antagonist in animal model of moderate brain injury by lateral fluid percussion.
All protocols conformed to the NIH Guide for care and use of laboratory animals and were approved by the Animal Experiment Committee of Tokyo Medical and Dental University. Measures were taken to reduce the number of animal used and their sufferings.
Animal and Housing
Adult Mongolian Gerbils of 24-36 weeks, weighing 60-100 gram were housed in groups of three or four and maintained on a 14/10 hours light/dark cycle with unlimited access to food and water.
Surgical Procedure for FPI
Brain injury was induced with fluid percussion injury (FPI) device as described previously  . In short, each animal was anesthetized with Sodium Pentobarbital (50 mg/kg i.p.), supplemented as necessary. The animals were allowed to breath spontaneously throughout the procedure. Once surgical level of anesthesia was achieved, the animal head was shaved, washed with iodine. Incision site was infiltrated with 1% lidocaine hydrochloride, animal was placed in a stereotaxic frame, an incision was made along the midline of scalp, a round craniotomy (3.5 mm diameter) was made on the right parietal bone with the center coordinating midway between bregma and lambda and 2.5 mm lateral to the midline. A 10 cm long firm silicone tube with an inner diameter of 2 mm attached to a fluid percussion injury device (Dragonfly R&D Inc.) was placed over the intact dura, bonded to the skull with cement glue. Moderate FPI was induced with a pressure of 26-32 psi (pound per square inch). Sham animals received anesthesia and underwent similar surgical procedure except for the delivery of fluid percussion. Following injury, the tube was removed from the skull and the scalp was sutured with nylon.
In the first stage, medication groups (trauma-medication n=9, sham-medication n=7) received intraperitoneal injection of SCH58261 at a dose of 0.01mg/kg body weight at 5 minutes after the surgery, then at 6 hours, then 12 hourly till day 5. Saline groups (trauma-saline n=7, sham-saline n=8) received same amount of saline for same duration.
In the second stage, the same dose of SCH58261 was injected intraperitoneally in the injured animal for different duration, namely single dose (n=6), 5 days (n=9) and 9 days (n=5) dose.
Open field test
We used the open field test to evaluate spontaneous locomotor activity as described previously  . Animals were placed individually in an open field apparatus (85 cm X 85 cm at the bottom). All the movements were tracked and measured for 10 min by a video tracking system and smart software (Bio Research Center, Nagoya, Japan). We then analyzed the total distance moved (cm/10 min) by each animal, which represents spontaneous locomotor activity.
A 40 gram net whose rods were 3mm in diameter (suitable for the gerbils’ grip)  was used in this study. Each gerbil was passed over the net, and lifted by its tail. The time the gerbils held the net freely was counted and used for evaluation. 6 trials were applied with 15 minutes interval. Mean time was counted.
In vitro autoradiography
After carrying out the behavior test on post-trauma day 9, the animals were sacrificed under ether anesthesia, the brains were rapidly removed and frozen. 20 µm thick coronal slices were dissected using a cryotome at -10º C (Bright Instrument, Huntingdon, UK). The brain slices were mounted on glass slides, dried and stored under -84º C until they were taken out for autoradiography.
In vitro autoradiography of the brain sections was performed similar to the procedure described before . In short, frozen sections of the brain were preincubated in a solution of 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA and 10 mM MgCl2, at 25ºC for 10min. [3H]ZM241385 (20 nmol/mL), 1U/mL Adenosine deamimase was added to the incubation solution, and the brain sections were incubated at 25ºC for 90 min. [3H]ZM241385 is an A2A receptor antagonistic radioligand in the rat brain. After incubation, these sections were washed twice for 2 min with cold buffer, dipped in cold distilled water, and dried with cold air. These brain sections were placed in contact with imaging plates (BAS-MS2025, Fujifilm, Tokyo, Japan), and regional brain radioactivity was analyzed using a bioimaging analyzer.
Regions of interests (ROIs) were placed over several brain regions with a reference brain atlas. Radioactivity on the brain regions were quantified using an image analysis software (Multi Gauge version 2.3, Fujifilm) and expressed as photostimulated luminescence (PSL)/area (mm2).
Fig. 1 Open field test: Moderate traumatic brain injury increased the mean total distance moved by animal in 10 min on all post traumatic days (open square). SCH58261 reduced the hyperlocomotion (closed square) to the level of sham groups. SCH58261 did not make any significant change in locomotion in sham group. All data presented as Mean±SEMFigure 1
Fig. 2 Representative movement track of the animal from each group: moderate lateral fluid percussion injury caused hyperlocomotion. The track of the trauma-medication group was similar to that of the sham groups.
Movement track of representative animal from each group on different days in the open field test is shown in fig. 2. Track of the animal in TBI group shows higher total distance moved in 10 min.
Fig. 3 Grabbing test: trauma-saline group grabbed the 40gm weight for significantly longer time on 3-9 post trauma days compared to sham groups. SCH58261 kept this grabbing time to the level similar to sham groups.
In fig. 3 animals in the TBI group grabbed the 40gm weight for significantly longer time on days 3-9 post-trauma days compared to sham groups (p<0.05). In sham groups the grabbing time did not change over the whole experiment period. In Trauma-Medication group, SCH58261 kept this grabbing time to the level similar to sham groups until day 7 post-trauma.
Duration of treatment
Fig. 4 Comparison among effects of SCH58261 for different duration of administration. Administration of single dose of SCH58261 ameliorated the hyperlocomotion up to 5th post trauma day (open square). Administration of SCH58261 for 5 or 9 days (closed triangle and closed circle, respectively) significantly reduced the locomotion to the level of sham groups during the whole observation period. (For clear view, the trauma and sham groups are not shown in this figure).
In the 2nd stage of the experiment (fig. 4), administration of single dose of SCH58261 kept the locomotion significantly lower until 5th post-trauma day after which the animals began to show increasing locomotion and on 7th day the locomotion in the TBI animals was higher than pre-injury level. Injection of SCH58261 for 5 or 9 days kept the locomotion significantly lower during the whole observation period of 9 days.
In vitro autoradiography
Fig. 5 Autoradiography using an A2A receptor antagonistic radioligand [3H]ZM241385. No significant difference in radioactivity was detected in the brain sections of the different groups.
In the autoradiography (Fig. 5), there was no significant difference in radioactivity of antagonistic radioligand in the A2A receptor binding, either in the cerebrum or in the striatum among the groups.
In this study, we looked for the effect of a selective A2A receptor antagonist, SCH58261 on the post-traumatic hyperlocomotion in Mongolian gerbils.
FPI in Mongolian Gerbils
For inducing generalized TBI, fluid percussion injury (FPI) and controlled cortical impact (CCI) have been used widely. Mongolian gerbil can be used for predictable pathological and behavioral outcomes in TBI  . In our laboratory we used reproducible models of moderate TBI by lateral fluid percussion injury in Mongolian gerbils.
Post TBI cognitive dysfunction and hyperlocomotion
Traumatic Brain Injury (TBI) causes a spectrum of physical, neurological and emotional/behavioral impairments, which can last for a long period. The primary impact of TBI, leads to mechanical events such as laceration, contusion, shearing and axonal stretching. Secondary injuries, which result from complex biochemical and physiological pathways, develop over hours to days and substantially contribute to chronic neurological symptoms . Clinical studies have correlated many of the TBI-induced symptoms with cognitive dysfunction and memory impairment . Post-traumatic cognitive dysfunction and hyperactivity results from number of mechanisms related to TBI such as, selective neuronal death in hippocampus , diffuse axonal injury . In TBI the duration of the hyperlocomotion was related to the degree of injury  .
In our study, we induced moderate TBI by FPI at 26-32 psi. In the open field test (fig. 1), the gerbils showed hyperlocomotion for the entire duration of the observation period of 9 days which is characteristic of the moderate TBI.
Effect of adenosine in TBI and other brain insult
Adenosine is thought to play role in many of the secondary damages following brain insults. Its level rises markedly in response to ischemia, hypoxia, excitotoxiciy, inflammation and other brain insults . TBI causes an increase in the intracerebral adenosine level  , which is also detectable in cerebrospinal fluid  . Raised level of adenosine after TBI has shown correlation with high mortality . The intracerebral raised adenosine in TBI is associated with the metabolic failure  . Adenosine neuromodulation alters the concentration of other neurotransmitter such as Glutamate, GABA, aspartae, taurine and other excitatory amino acids  .
Adenosine receptors in brain, neuromodulation and A2A receptor antagonist
Adenosine acts through multiple G-protein coupled receptors (Subtypes A1, A2a, A2B, A3) to exert a variety of physiological effects. Adenosine A3 receptor agonist reduces ischemia induced brain injury in rodents . In some ischemia models, A1 receptor agonists reduced cerebral infarction and improved the neurobehavioral outcome . In TBI A1 receptor agonists improved motor function and a better CA3 hippocampal cell count in cortical impact trauma model mice . Pretreatment with caffeine showed improved outcome in trauma model of mice with correlated upregulation of brain A1 receptor mRNA and inhibition of glutamate release . On the other hand, CGS21680, an A2A receptor agonist caused delay in recovery from traumatic brain injury. In ischemia model of rat, Adenosine A2A receptor antagonist SCH 58261  has shown improved outcome, reduced cortical damage and reduced ischemia induced outflow of excitatory amino acids, GABA, and adenosine from the striatum. Another A2A receptor antagonist CGS15943 was reported to significantly reduce cortical glutamate outflow after ischemia induced by four–vessel occlusion . In A2A receptor knocked out mice, the outcome was better in after TBI  with a reduced intracerebral glumate level . A2A receptor has potential to play a significant role in the neurotransmitter interaction, excitatory effect and in the outcome after traumatic brain injury.
In our study, the intraperitoneal administration of SCH 58261, an A2A receptor antagonist, (at the dose of 0.01 mg/kg body weight) reduced the hyperlocomotion seen after the TBI. A single dose administration was effective till the 5th post-trauma day, while continuous administration of the drug (at least for 5 days) kept locomotion near to the non-injured animal for the entire observation period of 9 days. There was no visible difference between administering the drug for 5 days and 9 days.
A2A receptor in autoradiography
In a study of ischemia in rats, adenosine A1 receptor gene expression was noted to be increased . In our experiment, no significant difference was detected in the A2A receptor binding either in the striatum or in the cortex among the groups, as revealed by [3H]ZM241385 autoradiography.
SCH58261, adenosine A2A receptor antagonist, reduced the hyperlocomotion and hyperactivity seen after traumatic brain injury. Effect of continuous administration was prolonged compared to single administration of the drug. SCH58261 may possess potential to improve cognitive dysfunction seen after traumatic brain injury.
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Saad Habib-E-Rasul Mullah1 , Jahanara Urmy2, Motoki Inaji1, Tadashi Nariai1, Satoru Ishibashi3, Kikuo Ohno1
1. Department of Neurosurgery, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan.
2. Dhaka Medical College, Dhaka, Bangladesh.
3. Department of Neurology, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan
Corresponding Author: Saad Habib-E-Rasul Mullah.
Current affiliation of corresponding author: NeuroTrauma Department, Naval Medical Research Center, Maryland, USA.
Address: 503 Robert Grant Avenue, Silver Spring, MD 20910 USA
Tel: +1-301-319-7522; Fax: +1-301-319-7486
Traumatic brain injury (TBI), Fluid percussion injury (FPI), Adenosine, A2A receptor, Autoradiography.