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2017
Disease-modifying effect of atipamezole in a model of post-traumatic epilepsy
Nissinen J
Elsevier BV
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http://dx.doi.org/10.1016/j.eplepsyres.2017.07.005
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Accepted Manuscript
Title: Disease-modifying effect of atipamezole in a model of post-traumatic epilepsy
Authors: Jari Nissinen, Pedro Andrade, Teemu Natunen, Mikko Hiltunen, Tarja Malm, Katja Kanninen, Joana I.
Soares, Olena Shatillo, Jukka Sallinen, Xavier Ekolle Ndode-Ekane, Asla Pitk¨anen
PII: S0920-1211(17)30206-1
DOI: http://dx.doi.org/doi:10.1016/j.eplepsyres.2017.07.005
Reference: EPIRES 5768
To appear in: Epilepsy Research Received date: 11-4-2017
Revised date: 30-6-2017 Accepted date: 6-7-2017
Please cite this article as: Nissinen, Jari, Andrade, Pedro, Natunen, Teemu, Hiltunen, Mikko, Malm, Tarja, Kanninen, Katja, Soares, Joana I., Shatillo, Olena, Sallinen, Jukka, Ndode-Ekane, Xavier Ekolle, Pitk¨anen, Asla, Disease-modifying effect of atipamezole in a model of post-traumatic epilepsy.Epilepsy Research http://dx.doi.org/10.1016/j.eplepsyres.2017.07.005
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Disease-modifying effect of atipamezole in a model of post-traumatic epilepsy
Jari Nissinen1, Pedro Andrade1, Teemu Natunen2, Mikko Hiltunen2, Tarja Malm1, Katja Kanninen1, Joana I Soares3, Olena Shatillo1, Jukka Sallinen4, Xavier Ndode-Ekane1, Asla Pitkänen1
1Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland
2Institute of Biomedicine, University of Eastern Finland and Department of Neurology, Kuopio University Hospital, PO Box 1627, FI-70211 Kuopio, Finland
3Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal; Programa Doutoral em Neurociências, Universidade do Porto, Porto, Portugal.
4Orion Corporation ORION PHARMA, P O Box 425 (Tengströminkatu 8), 20101 Turku, Finland.
Corresponding author: Asla Pitkänen, MD, PhD, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland, Tel: +358- 50-517 2091, Fax: +358-17-16 3030, E-mail: asla.pitkanen@uef.fi
Number of Words in Abstract: 365 Number of Figures: 6
Number of Tables: 7
Number of Supplementary Figures: 6
2 Highlights
Atipamezole, improved motor performance after traumatic brain injury (TBI)
Atipamezole reduced seizure susceptibility after TBI
Atipamezole had no effect on spatial memory performance after TBI
SR141716A had no-disease modifying effect on TBI outcome
Abstract
Treatment of TBI remains a major unmet medical need, with 2.5 million new cases of traumatic brain injury (TBI) each year in Europe and 1.5 million in the USA. This single-center proof-of-concept preclinical study tested the hypothesis that pharmacologic neurostimulation with proconvulsants, either atipamezole, a selective α2-adrenoceptor antagonist, or the cannabinoid receptor 1 antagonist SR141716A, as monotherapy would improve functional recovery after TBI. A total of 404 adult Sprague- Dawley male rats were randomized into two groups: sham-injured or lateral fluid-percussion–induced TBI. The rats were treated with atipamezole (started at 30 min or 7 d after TBI) or SR141716 (2 min or 30 min post-TBI) for up to 9 wk. Total follow-up time was 14 wk after treatment initiation. Outcome measures included motor (composite neuroscore, beam-walking) and cognitive performance (Morris water-maze), seizure susceptibility, spontaneous seizures, and cortical and hippocampal pathology. All injured rats exhibited similar impairment in the neuroscore and beam-walking tests at 2 d post-TBI.
Atipamezole treatment initiated at either 30 min or 7 d post-TBI and continued for 9 wk via subcutaneous osmotic minipumps improved performance in both the neuroscore and beam-walking tests, but not in the Morris water-maze spatial learning and memory test. Atipamezole treatment initiated at 1 wk post-TBI also reduced seizure susceptibility in the pentylenetetrazol test 14 wk after treatment initiation, although it did not prevent the development of epilepsy. SR141716A administered as a single dose at 2 min post-TBI or initiated at 30 min post-TBI and continued for 9 wk had no recovery- enhancing or antiepileptogenic effects. Mechanistic studies to assess the α2-adrenoceptor subtype specificity of the disease-modifying effects of atipametzole revealed that genetic ablation of α2A- noradrenergic receptor function in Adra2A mice carrying an N79P point mutation had antiepileptogenic effects after TBI. On the other hand, blockade of α2C-adrenoceptors using the receptor subtype-specific
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antagonist ORM 12741 had no favorable effects on the post-TBI outcome. Finally, to assess whether regulation of the post-injury inflammatory response by atipametzole in glial cells contributed to a favorable outcome, we investigated the effect of atipamezole on spontaneous and/or lipopolysaccharide-stimulated astroglial or microglial cytokine release in vitro. We observed no effect.
Our data demonstrate that a 9-wk administration of α2A-noradrenergic antagonist, atipamezole, is recovery-enhancing after TBI.
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Key words: α2-adrenoceptor – beam walking – cannabinoid 1 receptor antagonist - composite neuroscore - epileptogenesis – lateral fluid-percussion - memory – seizure susceptibility - somato- motor performance – SR141716A
1. Introduction
Traumatic brain injury (TBI) affects 2.5 million people annually in Europe (Corrigan et al., 2010;
Peeters et al., 2015). Almost half of the injured patients develop life-compromising functional impairments, including somatomotor and cognitive disabilities, and epilepsy (Corrigan et al., 2010).
Importantly, recovery from disabilities and epileptogenesis can continue in parallel for months to years.
Katz et al (Katz et al., 1998) reported that the recovery of upper arm motor functions occurs largely within the 6 months post-TBI in 82% of patients, whereas Hammond et al (Hammond et al., 2004) reported that cognitive recovery continues for up to 5 y. On the other hand, Haltiner et al (Haltiner et al., 1997) demonstrated that ~80% of those who develop epilepsy within a 5-y post-TBI follow-up will exhibit unprovoked seizures within 2 years. Thus, the neuronal and network plasticity underlying both motor and cognitive recovery as well as epileptogenesis progresses within the same time window, suggesting mechanistic convergence. Despite a large number of preclinical and clinical studies, no disease-modifying pharmacotherapies to prevent or alleviate post-TBI morbidity are yet on the market (Marklund and Hillered, 2011; Diaz-Arrastia et al., 2014).
TBI results in long-lasting noradrenergic hypofunction (Levin et al., 1994). Further, selective lesions of central noradrenergic pathways impair post-TBI recovery, and drugs that deplete noradrenaline levels, block 1 receptors, or decrease noradrenaline release (2 agonists) impede recovery (reviewed by Goldstein, 1999). Drugs that increase noradrenaline release (2 antagonists), however, facilitate recovery (Goldstein, 1999). These and many other studies led to the noradrenergic hypothesis of recovery, i.e., that increased efferent output from the locus coeruleus will enhance recovery, while reduced output from the locus coeruleus will retard recovery (Feeney and Hovda, 1985;
Goldstein and Bullman, 1997).
Atipamezole (ATI) is a selective 2-adrenergic antagonist that has a high 2/1 adrenoceptor selectivity ratio but does not display differential affinity for 2-adrenoceptor subtypes (Haapalinna et al., 1997; Virtanen et al., 1989). ATI rapidly penetrates the blood-brain-barrier (Biegon et al., 1992) and increases the release of noradrenaline (Gobert et al., 1997; Laitinen et al., 1995). A net effect of 2
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blockade is increased responsiveness of locus coeruleus noradrenergic neurons to stimulation (Sara and Bergis, 1991). Considering that 2-receptors are located in noradrenergic terminals (autoreceptors) as well as in dopaminergic and serotonergic terminals, the effects of ATI are not likely confined to the noradrenergic system. Moreover, previous studies demonstrated that ATI treatment increases motor recovery after medial cerebral artery stroke in rats as assessed by a limb-placing test (Puurunen et al., 2001), and the effect is associated with normalization of [14C]deoxyglucose uptake (Barbelivien et al., 2002). Our previous studies indicated that a 9-wk ATI treatment initiated at 1 wk after the induction of status epilepticus (SE) remarkably reduces seizure frequency (Pitkänen et al., 2004). The beneficial effect of ATI on the epileptogenic process is surprising as ATI also acts as a proconvulsant – i.e., it potentiates kainate acid-induced convulsions and increases mortality after SE (Halonen et al., 1995).
Based on these previous observations, we hypothesized that rather than depressing neuronal activity, enhancing excitability via pharmacologic neurostimulation using ATI will enhance post-TBI functional recovery and prevent the epileptogenic process. Further, the recovery-enhancing effect is not dependent on the mechanisms of action of the proconvulsant used. Consequently, the present study addressed the following questions: (1) Can motor and cognitive recovery after TBI induced by lateral fluid-percussion be improved by pharmacologic neurostimulation using a proconvulsant compound that blocks 2-adrenergic receptors (ATI)? (2) Does blockade of 2-adrenergic receptors prevent the development of a lowered seizure threshold and epilepsy? (3) Does the timing of ATI treatment initiation, 30 min or 7 d post-TBI, affect the outcome? To investigate, whether the expected favourable effects on recovery would be specific to 2-adrenergic receptor blockage, we also assessed the effect of another neurostimulant, the cannabinoid receptor 1 (CB1) antagonist SR141716A (Rimonabant®), on post-TBI recovery and epileptogenesis. Selection of SR141716A as a comparator treatment was based on previous observations of Echegoyen et al. (2009) and Wang et al. (2016) who reported its antiepileptogenic effects after TBI.
Our findings demonstrated that ATI treatment initiated after TBI both enhanced somato-motor recovery and reduced seizure susceptibility. Administration of SR141716A had no favorable effects on outcome.
6 2. Materials and Methods
Figure 1 shows the design of this statistically powered, randomized, single-center, vehicle- controlled proof-of-concept study.
All animal procedures were approved by the Animal Ethics Committee of the Provincial Government of Southern Finland, and performed in accordance with the guidelines of the European Community Council Directives 2010/63/EU.
2.1. Animals and induction of lateral fluid-percussion injury
Male Sprague-Dawley rats (Harlan, The Netherlands) were 12 wk old at the time of TBI. Details of the environment, housing, and diet are summarized in Table 1.
Severe lateral fluid-percussion injury (FPI) was induced as described previously (Kharatishvili et al., 2006). Briefly, rats were anesthetized (6 ml/kg, i.p.) with a mixture containing sodium pentobarbital (58 mg/kg), chloral hydrate (60 mg/kg), magnesium sulfate (127 mg/kg), propyleneglycol (43%), and ethanol (11.7%), and then placed into a stereotaxic frame with lambda and bregma at the same horizontal level. A midline scalp incision was made and a 5-mm craniotomy was centered over the left cortex, midway between the lambda and bregma, and midway between the sagittal suture and temporal ridge. The dura was left intact and a plastic female Luer-Lock connector was secured in the craniotomy with Vetbond adhesive (3M, St.Paul, MN, USA). The connector was anchored with dental acrylate to a screw placed in the skull rostral to the bregma. Animals were placed on heating pads while anesthetized to maintain normothermic temperature. Ninety minutes after injection of the anesthetic, the rat was attached to the fluid-percussion device (Amscien Instruments, Richmond, Virginia, USA) to produce TBI (pressure level adjusted to 3.0 atm). Occurrence of immediate post-impact behavioural seizures and duration of apnea were monitored for 5 minutes. Animals were removed from the device, and then the dental cement, screw, and Luer-Lock connector were removed, and the scalp was sutured.
Sham animals underwent surgery, but were not injured.
2.2. Randomization of animals into different treatment groups
A total of 404 rats were randomized by lottery into 16 different treatment groups in 4 treatment arms, assessing two treatments (ATI, SR141716A) and two time windows (Fig. 2). The animal numbers were estimated based on power calculations that were tuned to detect epileptogenesis after lateral FPI. Based on four previous independent experiments, the latency of the TBI group to the 1st spike in
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the pentylenetetrazol (PTZ)-test was decreased to 42% that of controls. We assumed that the latency to the 1st spike would be 90% that in controls. Thus, we would need 30 drug-treated TBI rats, if the vehicle group had 13 injured animals (MedCalc software, Δ 0.48, SD 0.5, power 0.8, p<0.05). Our preliminary data indicated that these animal numbers would be sufficient to detect a 3-point difference in the composite neuroscore between drug-treated and vehicle-treated TBI animals (MedCalc software, power 0.80, p<0.05). Moreover, based on previous studies, we expected a 25% acute (<72 h) and 15%
follow-up mortality or exclusion for other reasons (e.g., loss of electrode headset)(Kharatishvili et al., 2006).
2.3. Drugs and Preparation of Treatments
Atipamezole. ATI was a kind gift from Orion Pharma (Finland). Due to its short half-life of elimination (1.3 h in rats after intraperitoneal administration; Orion Pharma, unpublished observations), ATI was delivered via subcutaneous Alzet minipumps to maintain a constant therapeutic drug level in the brain during the chronic experiment (Pitkänen et al., 2004). The dose used was the same as in previous studies, in which ATI administration exhibited a disease-modifying effect on post-stroke motor recovery and SE-induced epileptogenesis (Pitkänen et al., 2004; Puurunen et al., 2001). Briefly, Minipumps (2ML1 and 2ML4, Durect Corporation, CA, USA) were activated at +37oC in 0.9% NaCl and implanted subcutaneously into the back between the scapulae of the anesthetized rat, according to the instructions provided by manufacturer. Atipamezole was dissolved in 0.9% NaCl and its concentration was adjusted according to the weight of the rat. The pumping rate (10 µl/h in a 1-wk pump and 2.5 µl/h in a 4-wk pump) was adjusted to 100 µg of atipamezole (ATI)/kg/h. Delivery of atipamezole from the minipumps was confirmed by measuring the volume of the remaning solution after pump removal. The brain and plasma levels of atipamezole, resulting from the administration scheme used here, have been reported previously (Pitkänen et al., 2004).
SR141716A. SR141716A was purchased from AK Scientific, Inc. (Union City, CA, USA). Based on previous positive data (Chen et al., 2007; Echegoyen et al., 2009), SR141716A was administered intraperitoneally once per day at a dose of 10 mg/kg. For the TBI-SR141716A 2 min group (single dose administration), SR141716A was dissolved in Tween 80. For the TBI-SR141716A 30 min group, SR141716A was emulsified by sonication in 1% Tween 80 made in sterile 0.9 % NaCl to provide a better tolerated vehicle for chronic use.
8 2.4. Treatment groups
ATI treatment was initiated at either 30 min or 7 d post-TBI and continued for 9 wk. SR141716A treatment was initiated as either a single dose at 2 min to reproduce the data by Echegoyen et al.
(2009), or at 30 min post-TBI and continued for 9 wk (Fig. 1).
2.4.1. Atipamezole 30 min treatment arm
Sham-NaCl 30 min: Rats were treated with a single injection of 0.9 % NaCl (2 ml/kg, i.p.) at 30 min after sham-operation. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with 0.9% NaCl was continued for 9 wk.
Sham-ATI 30 min: Rats were treated with a single injection of ATI (1 mg/kg, 2 ml 0.9% NaCl/kg, i.p.) at 30 min after sham-operation. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with ATI (100 μg/kg/h) was continued for 9 wk.
TBI-NaCl 30 min: Rats were treated with a single injection of 0.9 % NaCl (2 ml/kg, i.p.) at 30 min after TBI. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with 0.9% NaCl was continued for 9 wk.
TBI-ATI 30 min: Rats were treated with a single injection of ATI (1 mg/kg, 2 ml 0.9% NaCl/kg, i.p.) at 30 min after TBI. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with ATI (100 μg/kg/h) was continued for 9 wk.
2.4.2. Atipamezole 7 d treatment arm
Sham-NaCl 7 d: Rats were treated with a single injection of 0.9% NaCl (2 ml/kg, i.p) at 7 d after sham- operation. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with 0.9%
NaCl was continued for 9 wk.
Sham-ATI 7 d: Rats were treated with a single injection of ATI (1 mg/kg, 2 ml 0.9% NaCl/kg, i.p.) at 7 d after sham-operation. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with ATI (100 μg/kg/h) was continued for 9 wk.
TBI-NaCl 7 d: Rats were treated with a single injection of 0.9 % NaCl (2 ml/kg, i.p.) at 7 d after TBI.
Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with 0.9% NaCl was continued for 9 wk.
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TBI-ATI 7 d: Rats were treated with a single injection of ATI (1 mg/kg, 2 ml 0.9% NaCl/kg, i.p.) at 7 d after TBI. Alzet minipumps (s.c.) were implanted at the same session, and chronic treatment with ATI (100 μg/kg/h) was continued for 9 wk.
2.4.3. SR141716A 2 min treatment arm
Sham-Tween 2 min: Rats were treated with a single dose of Tween 80 (2 ml/kg) at 2 min after sham- operation. No further treatment was administered.
Sham-SR141716A 2 min: Rats were treated with a single injection of SR141716A (10 mg/kg, diluted in Tween 80, 2 ml/kg) at 2 min after sham-operation. No further treatment was administered.
TBI-Tween 2 min: Rats were treated with a single injection of Tween 80 (2 ml/kg) at 2 min after TBI.
No further treatment was administered.
TBI-SR141716A 2 min: Rats were treated with a single injection of SR141716A (10 mg/kg, diluted in Tween 80, 2 ml/kg, i.p.) at 2 min after TBI. No further treatment was administered.
2.4.4. SR141716A 30 min treatment arm
Sham-Tween 30 min: Rats were treated with a single injection of Tween 80 (2 ml/kg, i.p.) at 30 min after sham-operation. Thereafter, treatment was continued with 1% Tween 80 (diluted in 0.9 % NaCl, 2 ml/kg, i.p.) once per day for 9 wk.
Sham-SR141716A 30 min: Rats were treated with a single injection of SR141716A at 30 min after sham-operation (10 mg/kg, diluted in Tween 80, 2 ml/kg). Thereafter, treatment was continued with SR141716A (10 mg/kg/d, in 1% Tween diluted in 0.9% NaCl, 2 ml/kg, i.p.) once per day for 9 wk.
TBI-Tween 30 min: Rats were treated with a single injection of Tween 80 (2 ml/kg, i.p.) at 30 min after TBI. Thereafter, treatment was continued with 1% Tween 80 (diluted in 0.9% NaCl, 2 ml/kg, i.p.) once per day for 9 wk.
TBI- SR141716A 30 min: Rats were treated with a single injection of SR141716A at 30 min after TBI (10 mg/kg, dissolved in Tween 80, 2 ml/kg, i.p.). Thereafter, treatment was continued with SR141716A (10 mg/kg/d, in 1% Tween diluted in 0.9% NaCl, 2 ml/kg, i.p) once per day for 9 wk.
2.5. Assessment of Outcome measures
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As primary outcome measures, we analyzed (1) behavioral (composite neuroscore, beam- walking) and cognitive (Morris water-maze) recovery, (2) epileptogenesis [spontaneous electrographic seizures, handling-related seizures, and seizure susceptibility in the PTZ-test performed under continuous video-electroencephalography (EEG)], and (3) structural outcome (cortical lesion volume, mossy fiber sprouting) according to the schedule presented in Fig. 1.
2.5.1. Composite neuromotor score
The neuroscore is a sensitive indicator of the effect of pharmacologic manipulations (McIntosh et al., 1989; Okiyama et al., 1992). Testing was performed at 1 d before, at 2 d and 7 d post-TBI, and thereafter at 2, 3, 4, 5, 6, 7, 8, and 9 wk post-TBI, as well as at the end of a 2-wk washout period. Animals were scored from 0 (severely impaired) to 4 (normal) for each of the following 7 indices: (a) left and right (2 indices) forelimb flexion during tail suspension, (b) left and right (2 indices) hindlimb flexion when the forelimbs remained on a hard surface and the hindlimbs were lifted up and back by the tail, (c) ability to resist a lateral pulsion toward the left and right (2 indices), and (d) angle board.
Contraflexion test-forelimb test: To elicit a reflex, the animal was lifted by the tail and lowered head down toward the mat. Forelimb response/motions were monitored as this procedure was repeated 2-3 times. Animals received separate score for the left and right forelimbs. Scoring was done as follows: Score 0 (most impaired) = no response; nose hit the mat. Score 1 = some response; arms were extended and remained mainly perpendicular to the plane of the rat's body when suspended;
limbs displayed spasticity. Score 2 = some limb spasms; arms were extended either perpendicular or parallel to body plane. Score 3 = arms were completely extended forward, but the response lacked strength. Score 4 (normal) = instant response; arms were extended forward in a smooth fluid motion, but there was a crook in the forelimbs as they extended.
Hindlimb flexion test: The animal’s eyes were shielded with a cupped hand while performing test.
The animal was pulled back gently and quickly from its tail, and the hindlimbs observed. Separate scores were given for the left and right legs. Scoring was done as follows: Score 0 = no response. Score 1 = some response, limb flipped back but did not extend. Score 2 = some limb extension, feet flipped back but lacked strength. Score 3 = hindlimbs extended back fully and flipped back fully as well, but motion lacked strength and quality. Score 4 = instant response; hindlimbs shot back and out (limbs extended, feet flipped back, and toes spread).
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Lateral pulsion test: The animal was pushed from its side across a vertically grooved rubber mat on the table to test its strength of resistance to pulsion. The animal was placed longitudinally, facing away from the observer. The animal was first pushed to the left and then to the right. Thereafter, the observer tried to flip the animal over, first to the left and then to the right. Scoring was done as follows:
Score 0 = rat offered no resistance, it rolled over and did not righten itself. Score 1 = rat offered minimal resistance before rolling over, then rolled over but rightened itself slowly. Score 2 = rat did not roll over and offered minimal resistance. Score 3 = rat offered strong resistance, but observer could still push it across the mat, rat did not roll over. Score 4 = rat exhibited strong resistance, gripped the mat strongly, and/or moved legs in a coordinated manner when the observer pushed it across the mat, but the observer had to exert effort to move the rat across the mat.
Angle board test: Finally, the animals were tested for their ability to stand on an inclined plane.
An angle board (50 x 60 cm) was covered with a vertically grooved rubber mat. To obtain a baseline value, the test was started by positioning the board at 40° angle. The animal was placed on the board and first tested in the vertical direction, then left, and then right. The animal had to stand still for 5 s without holding on by its tail to get credit for the angle. Each animal was given three attempts in each direction before the angle was increased. The angle was increased by 2.5° each time until the rat could no longer stand on the board. The maximum angle was scored. In post-injury testing, animals started at 10° below their base-line value. Scores (0-4) were assigned for each direction according to their difference from pre-injury performance, and then averaged for inclusion in the composite score.
Scoring was done as follows: every 2.5° decrease from the baseline angle equaled a 1 point reduction, starting from 4. Score 0 = there was a 10° decrease or more from the baseline. Score 1 = a 7.5° decrease from the baseline. Score 2 = a 5° decrease from the baseline. Score 3 = a 2.5° decrease from the baseline. Score 4 = no change or angle was higher compared to baseline.
A composite neuroscore (0-28) was generated by summing the scores from each of the 7 tests.
2.5.2. Beam-walking
To evaluate complex motor movement and coordination, beam-walking was performed at 1 d before, at 2 d and 7 d post-TBI, and thereafter at 2, 3, 4, 5, 6, 7, 8, and 9 wk after TBI, and 3-6 h after the last administration of vehicle, ATI, or SR-141716A (Ohlsson and Johansson, 1995). Testing was also performed at the end of 2-wk washout period (i.e., 74 d after TBI).
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The beam, a 1390-mm long and 21-mm wide wooden bar, was placed 430mm above the floor.
At the right end of the beam, there was a black box (250 x 200 mm). A wall was placed 30 cm to theleft of the beam. A mirror was placed behind the beam on the side wall. Starting 2 d before TBI, rats were habituated to the beam. Animals were placed into the box for 1 min. Thereafter, the animal was placed on the beam at a starting distance of 15 cm from the box. The animal was allowed to go the box and remain in the box for 1 min. Thereafter, the animal was placed on the beam at a starting distance of 35 cm from the box. The animal was allowed to go into the box and stay there for 1 min. This step was then repeated. On the next day, the animal was placed into the box for 1 min, and allowed to enter the box starting at 35 cm, followed by 70 cm, and 100 cm distance from the box. On testing day, the animal was allowed to cross the whole beam three times, which was scored from 0 (rat falls down) to 6 (rat crossed the beam withno foot slips). Between each run, the animal was in the box for 1 min. Scoring was as follows:Score 0 = rat fell down. Score 1 = rat was unable to traverse thebeam, but remained sitting across the beam. Score 2 = rat fell downwhile walking. Score 3 = rat could traverse the beam, but the affectedhindlimb did not aid in forward locomotion. Score 4 = rat traversedthe beam with more than 50% foot slips. Score 5 = rat crossed thebeam with a few foot slips. Score 6 = rat crossed the beam withno foot slips. The mean score of three runs was calculated.
2.5.3. Morris water-maze
The spatial learning and memory performance of rats was tested in Morris water maze using a 3- d paradigm (Halonen et al., 1996). The time spent in the four quadrants of the maze was recorded. In addition, path length (swimming distance) and swimming speed were measured. Over a series of 10 trials on days 75 and 76 post-TBI (i.e., after treatment discontinuation), animals learned the location of a submerged platform using visual cues outside the maze. The rats were acclimated to the task on the first day of training. The time (latency) to reach the hidden platform was recorded for each trial. On day 77 post-TBI, the platform was removed from the maze apparatus and rats were allowed to swim for 60 s to evaluate their memory of the platform location (probe trial). The time spent in the four quadrants of the maze was recorded. In addition, path length (swimming distance) and swimming speed were measured.
2.5.4. Assessment of the development of epilepsy and seizure susceptibility
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Electrode implantation. Animals were anesthetized as described before and mounted in a stereotactic frame. Two stainless steel cortical screw electrodes (E363/20 Plastics One Inc., Roanoke, VA, USA) were placed over the parietal cortex (1 rostral to the craniectomy, 1 contralateral to the center of the craniectomy), and 2 into the skull bilaterally over the cerebellum to serve as a reference and ground electrodes. Electrodes were fixed to the skull using dental acrylic (Selectaplus, Dentsply, DeTrey GmbH, Dreieich, Germany).
Video-EEG monitoring. The first continuous video-EEG monitoring lasting for 1 wk was performed at the end of the drug-treatment period to detect the occurrence of seizures while the rats were still on proconvulsant treatments. The second video-EEG monitoring (for 2 wk) was performed after a 2-wk drug washout to observe seizure activity without proconvulsant medications.
For monitoring, rats were placed in Plexiglas cages (47x 29x 50 cm) where they could move freely (1 rat/cage). EEG was monitored (24/7) using the Nicolet One nEEG (ver. 5.71) recording system connected with a M40 (Taugagreining, Iceland) or Oxford (Medical Systems Division, UK) amplifier and filtered (high-pass filter 0.3 Hz cut-off, low-pass 100 Hz). The behavior of the animal was recorded using an IR-sensitive WV-BP330/GE Video Camera (Panasonic, wide angle-lens) that was positioned above the cages. EX12LED-3BD-9W infrared light (Bosch, Canada) was used at night to allow for continuous 24 h/d video monitoring.
Analysis of video-EEG. EEG files were analyzed manually by browsing through the recording on a computer screen (30 mm/s chart speed). EEG seizures were defined as high-amplitude rhythmic discharges that clearly represented a new pattern of tracing (repetitive spikes, spike-and-wave discharges, and slow waves) that lasted at least 5 s. Epileptic events occurring with an interval of less than 5 s without the EEG returning to baseline were defined as belonging to the same seizure. If an electrographic seizure was observed, its behavioral severity was scored according to a slightly modified Racine’s scale (Racine, 1972): score 0, electrographic seizure without any detectable motor manifestation; score 1, mouth and face clonus, head nodding; score 2, clonic jerks of one forelimb;
score 3, bilateral forelimb clonus; score 4, forelimb clonus and rearing; and score 5, forelimb clonus with rearing and falling. Seizures scored 0–2 were considered partial, whereas seizures scored 3–5 are considered secondarily generalized.
Electrographic seizures were verified in a blinded manner by two independent observers (PA and JN) who were not aware of the treatment of the animal.
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Handling related seizures. Some rats developed a seizure when handled. These occasions included angle board test, beam-walking, water maze, connection of EEG cable, injection of anesthetics, or temperature measurement. If the seizure occurred during behavioral testing, score of that day was not included in analysis. The severity of handling-related seizures was scored according to Racine (1972).
Pentylenetetrazol seizure susceptibility test. At the end of the second video-EEG monitoring period, seizure susceptibility was tested with a pentylenetetrazol (PTZ) test (Kharatishvili et al., 2006).
A sub-convulsant dose of PTZ (1,5-pentamethylenetetrazole, 98%, Sigma-Aldrich YA-Kemia Oy, Finland) was dissolved in 0.9% NaCl and injected i.p. (25mg/kg)(Kharatishvili et al., 2006). Following PTZ injection, rats were placed separately into transparent Plexiglas cages (47x 29x 50 cm) where they were able to move freely, and video-EEG was recorded from parietal electrodes for 24 h. Electrographic epileptiform discharge (ED) was defined as a high-amplitude rhythmic discharge containing a burst of slow waves, spike-wave and/or polyspike-wave components and lasting <5 s. A spike was defined as a high-amplitude (twice a baseline) sharply contoured waveform with a duration of 20–70 ms. Latency to the first spike, total number of spikes, latency to the 1st ED, total number of EDs, percentage of rats with induced seizures, duration of each induced seizure, and cumulative seizure duration were counted during 60 min after PTZ administration. Spike counting did not include the electrographic seizure events. In addition to continuous video recording, the behavior of the animal was monitored by an observer, and the severity of induced seizures was scored according to Racine (1972).
2.5.5. Histologic assessment of cortical and hippocampal damage and axonal plasticity Neuroprotection and axonal plasticity
After the 2nd video-EEG recording, rats were perfused for histology to assess the severity of cortical lesion (thionin) and axonal plasticity (mossy fiber sprouting). In addition, sections throughout the brain were assessed to detect pathologies that could result in non-TBI related epilepsy (particularly brain abscesses). A more detailed quantitative analysis focused on the ATI 7 d treatment arm, in which we found favorable effects on both the behavioral recovery and epileptogenesis.
Fixation. Anesthetized rats were intracardially perfused according to a Timm or 4%
paraformaldehyde fixation protocol (50:50 of rats for each fixation; for details see Ndode-Ekane et al., (2010). The brains were removed, cryoprotected in 20% glycerol in 0.02 M PB buffer, pH 7.4, for 24 h, frozen in dry ice, and stored at -70 oC. A 1-in-5 series of frozen coronal sections was cut (30 µm thick)
15
using a sliding microtome. Sections were collected into tissue collection solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium phosphate buffer) and stored at -20 oC until processed.
Staining and analysis of sections. One series of sections was stained with thionin to assess the severity of cortical atrophy and screen for other brain pathologies. Adjacent sections were Timm- stained and the intensity of mossy fiber sprouting was scored as described previously (Pitkänen et al., 2004). Severity of cortical damage was assessed using the Cavalieri estimation of cortical lesion volume as previously described (Kharatishvili and Pitkänen, 2010).
2.6. Follow-up of adverse events
The general well-being of the animals was monitored using a standardized form provided by the Animal Center, and body weight and rectal temperature were monitored once per week.
2.7. Exclusion criteria
Rats were excluded from the follow-up if body weight loss exceeded 20% of the initial weight, if there was a decrease in general well-being (e.g., cleaning of fur, abdominal swelling), or if the electrode headset was lost (one re-operation was performed if the skull was not damaged or infected). For the final data analysis, we included only animals for which histology was available and no signs of brain abscess or other dual pathologies were detected that could affect the structural and functional outcome.
2.8. Mechanistic analyses
To investigate the mechanisms of the favorable outcome observed in TBI rats treated with ATI, we performed the following experiments: (a) analysis of target expression by investigating the expression of noradrenergic α2A receptors in the brain at 2 d, 7 d, and 2 months after lateral fluid percussion injury with immunohistochemistry (Supplementary Fig. 1); (b) assessment of seizure susceptibility in mice carrying a D79N point mutation in α2A-adrenoceptor (Adra2A) at 2 months after controlled cortical impact–induced TBI to investigate whether genetic ablation of α2A-adrenoceptor function would mimic the effects of pharmacologic α2-adrenoceptor blockade (Supplementary Fig. 2);
(c) analysis of the effect of ATI on spontaneous and lipopolysaccharide-induced cytokine release in astrocytes (Supplementary Fig. 3); (d) analysis of the effect of ATI in neuronal/microglial cultures to assess the recently reported function of α2-adrenoceptors in peripheral and central neuroinflammation
16
(Birder and Perl, 1999; Herrera-García et al., 2014; Piwnica et al., 2014; Sukegawa et al., 2014; Wang et al., 2014) (Supplementary Figs. 4-5); and (e) analysis of the effect of the α2C-adrenoreceptor antagonist ORM-12741 (Herrick et al., 2014) on behavioral and cognitive performance, and epileptogenesis after experimental TBI in a study-design similar to that used in the present study (treatment was initiated at 30 min after TBI) (Supplementary Fig. 6).
2.9. Statistical analysis
Analysis of coded raw data was conducted in a blinded fashion to eliminate analysis bias.
Statistical analysis was performed using SPSS for Windows (v. 21). Within time-point comparisons between animal groups were tested with the non-parametric Kruskal-Wallis test followed by a post hoc analysis with the Mann-Whitney U test (composite neuroscore, beam-walking) or the 1-way ANOVA followed by Bonferroni post hoc analysis (as indicated in the text). Neuroscore and beam-walking were also analyzed using repeated measures ANOVA, followed by post hoc analysis with Dunnett’s test.
Differences in mortality as well as in the occurrence of early seizures, induced seizures, and epilepsy in different animal groups were tested with the 2-test. A p value <0.05 was considered significant.
17 3. Results
3.1. Impact pressure, time in post-TBI apnea, post-impact seizures, mortality, weight, rectal temperature, and exclusions
Impact pressure. Impact pressure used to induce TBI and time in post-injury apnea did not differ between treatment groups (Table 2). Immediate post-impact behavioral seizures were observed in 17%
(4/24) of rats in the TBI-NaCl 30 min, 26% (8/31) in the TBI-ATI 30 min, 23% (3/12) in the TBI-NaCl 7 d, 28% (8/29) in the TBI-ATI 7 d, 28% (5/18) in the TBI-Tween 2 min, 30% (10/33) in the TBI-SR141716A 2 min, 42% (5/12) in the TBI-Tween 30 min, and 27% (8/30) in the TBI-SR141716A 30 min groups (2, p>0.05). The mean duration of early seizures was 27 s (range 2-53 s), and it did not differ between treatment groups (2, p>0.05).
Mortality. Mortality in the different treatment groups is summarized in Table 3. Acute mortality (<72 h post-TBI) was 9% (7/78) in sham-injured animals and 17% (50/291) in rats with TBI (2, p>0.05).
In sham-injured animals, acute mortality was higher among rats randomized to the SR141716A treatment arm (6/36) than in those randomized to the ATI treatment arm (1/42; 17% vs. 2%; 2, p=0.044), due to the acute mortality in the Sham-SR141716A 2 min group (4/11, 36%). In TBI rats, acute mortality was comparable between the ATI (21/152) and SR141716A (29/139) treatment arms (14% vs.
21%; 2, p>0.05).
Follow-up mortality (>72 h post-TBI) was 18% (13/71) in sham-injured animals and 17% (40/241) in TBI rats (2, p>0.05)( Table 3). In sham-injured animals, follow-up mortality was comparable between the ATI (9/41) and SR141716A treatment arms (4/30; 22% vs. 13%; 2, p>0.05). In TBI rats, follow-up mortality was comparable between the TBI-NaCl (6/43, 30 min and 7 d groups combined) and TBI- Tween 30 min (0/15) groups (14% vs. 0%; 2, p>0.05). Follow-up mortality in chronically treated animals was higher in the TBI-ATI groups with Alzet minipumps (27/88, 30 min and 7 d groups combined) than in the TBI-SR141716A 30 min (2/36) group (31% vs. 6%; 2, p<0.01). Importantly, in the ATI treatment arm, follow-up mortality did not differ between the TBI-NaCl (6/43, 30 min and 7 d groups combined) and TBI-ATI (27/88) groups (14% vs. 28%; 2, p>0.05).
Body weight. Body weight of rats in the TBI-ATI 30 min group was 9% lower than that in the corresponding vehicle group at the 3-wk and 4-wk time-points (p<0.01) (Table 4). SR141716A did not affect the body weight (Table 4).
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Rectal temperature. Rectal temperature was not affected by ATI or SR141716A treatment (Table 5).
Exclusions. A flow-chart summarizing the number of animals at different stages of the study is shown in Fig. 1. In the end, we analyzed thionin-stained sections throughout the entire brain to identify non-TBI related brain pathologies that could relate to adverse outcomes. We found an abscess in the brain parenchyma in 15 rats, of which 5 were randomized to the ATI treatment arm (1 Sham-ATI 30 min, 1 to Sham-NaCl 7 d, 1 to TBI-NaCl 30 min, 1 to TBI-NaCl 7 d, 1 to TBI-ATI 7 d), and 10 were randomized to the SR141714A treatment arm (1 Sham-Tween 2 min, 1 TBI-Tween 2 min, 1 Sham- SR141716A 30 min, 3 TBI-Tween 30 min, 1 TBI-SR141716A 2 min, 3 TBI-SR141716A 30 min). These 15 rats were excluded from further analysis.
3.2. Atipamezole 30 min and 7 d treatment arms 3.2.1. Somatomotor and cognitive recovery
Composite neuroscore. The Kruskal-Wallis test revealed differences between groups in the composite neuroscore at each testing point over the course of the 9-wk follow-up (all time-points p<0.001). Both vehicle- and ATI-treated TBI groups performed poorly compared with the corresponding sham-injured groups (all time-points p<0.001). Post hoc analysis using the Mann-Whitney U test revealed that the TBI-ATI 30 min group performed better than the TBI-NaCl 30 min group, particularly during the 2nd month of treatment (Fig. 3A). Also, the TBI-ATI 7 d group performed better than the TBI- NaCl 7 d group (Fig. 3B).
Beam-walking. The Kruskal-Wallis test revealed differences between groups in the beam-walking test over the course of the 9-wk follow-up (p<0.01). Post hoc analysis with the Mann-Whitney U test revealed that the Sham-ATI 30 min group performed better than the Sham-NaCl group (Fig. 3C).
Importantly, the TBI-ATI 30 min group also performed better than the TBI-NaCl 30 min group (Fig. 3C), and did not differ from the Sham-NaCl 30 min group after only 1-wk ATI treatment (data not shown).
Also, the TBI-ATI 7 d group performed better than the TBI-NaCl 7 d group and reached the performance level of the Sham-NaCl 7 d group after only 1-wk ATI treatment (Fig. 3D). There was no difference in the performance between the 30 min or 7 d TBI-NaCl and the 30 min or 7 d TBI-ATI groups.
Morris water-maze. The Kruskal-Wallis analysis of the probe trial test on the 3rd testing day followed by the Mann-Whitney U test indicated that both vehicle- and ATI-treatment TBI groups in the ATI 30 min treatment arm were impaired compared with the corresponding sham-injured groups
19
(p<0.05). There was no difference between the vehicle- and ATI-treated injured groups in the 7-d treatment arm (data not shown).
3.2.2. Development of epilepsy
Rats were diagnosed with epilepsy if they exhibited either a spontaneous electrographic seizure or a handling-related seizure (Table 6; Fig. 4)(Fisher et al., 2014). Altogether, electrographic seizures were observed in 20 rats and handling-related seizures were observed in 17 rats. Of these animals, 6 had both electrographic and handling-related seizures. Handling-related seizures typically occurred while the rat was being connected to the video-EEG monitoring system or during beam-walking.
Spontaneous electrographic and/or handling-related seizures were observed in 9% (3/35) of injured rats in the TBI-NaCl groups (TBI-NaCl 30 min or TBI-NaCl 7 d groups combined; no difference between groups), which was expected based on our previous experience (Kharatishvili et al., 2006)(Table 6). ATI treatments initiated at either 30 min (26% had spontaneous seizures) or 7 d (21%) post-TBI did not prevent the development of epilepsy compared with the TBI-NaCl group (p>0.05, 2- test)(Table 6). There was no difference in the occurrence of spontaneous seizures between the 1st (animals still on ATI treatment) and 2nd EEG recordings (after washout), suggesting that ATI, a proconvulsant, did not provoke spontaneous seizures (2-test, data not shown). One of the rats in the Sham-NaCl 30 min group had one spontaneous seizure (duration 33 s). Histologic analysis of thionin- stained preparations revealed no structural abnormality, and therefore the rat was not excluded from the statistical analysis.
3.2.3. Development of seizure susceptibility
To assess whether the treatments reduced seizure susceptibility, animals were exposed to the PTZ seizure-susceptibility test at the end of the 2nd video-EEG monitoring period (4 wk after discontinuation of the treatment), as described before (Kharatishvili et al., 2006). High-quality EEGs from the PTZ test were available for 241 rats (54 sham-injured, 187 with TBI), of which 124 were from the ATI treatment arm (30 sham-injured, 94 with TBI). Overall, the occurrence of PTZ-induced seizures was higher in the TBI rats than in the sham-injured group (58% vs. 26%; p<0.001, 2test).
As there was no difference in parameters between the Sham-NaCl 30 min and 7 d groups, the data were combined for further statistical analysis (“Sham-NaCl All” group, Fig. 5). Similarly, data from the Sham-ATI 30 min or 7 d groups were combined (“Sham-ATI All” group, Fig. 5).
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Time to the 1st epileptiform spike. Kruskal-Wallis analysis revealed differences in the latency to the 1st spike between groups (p=0.028)(Fig. 5A). Post hoc analysis with the Mann-Whitney U test indicated that the latency to the 1st epileptiform spike was remarkably shorter in the TBI-ATI 30 min group than in sham-injured animals (Fig. 5A). ATI treatment initiated at 7 d post-TBI normalized the latency to the 1st epileptiform spike to the control level.
Number of epileptiform spikes during 60 min after PTZ administration. Kruskal-Wallis analysis revealed no differences between groups in the ATI treatment arm (p=0.242)(Fig. 5B).
Time to the 1st epileptiform discharge (ED) during 60 min after PTZ administration. Kruskal-Wallis analysis revealed no differences between groups in the ATI treatment arm (p=0.242)(Fig. 5C).
Number of EDs for 60 min after PTZ administration. Kruskal-Wallis analysis revealed a trend toward a difference between treatment groups (p=0.081). Post hoc analysis with the Mann-Whitney U-test revealed that the number of EDs was lower in the TBI-ATI 7 d group than in the TBI-NaCl (p=0.016) or TBI-ATI 30 min groups (p=0.023)(Fig. 5D).
Time to the 1st epileptiform event (either spike or ED). Kruskal-Wallis analysis showed a trend toward a difference in the latency to the 1st epileptiform event between groups (p=0.063). Post hoc analysis with the Mann-Whitney U test indicated that the latency to the 1st epileptiform event was remarkably shorter in the TBI-NaCl (p=0.023) and TBI-ATI 30 min groups (p=0.049) than in the sham- injured animals. ATI treatment initiated at 7 d post-TBI normalized the latency to the 1st epileptiform event to the control level (data not shown).
Time to 1st PTZ-induced seizure. Kruskal-Wallis analysis revealed no differences between groups in the ATI treatment arm (p=0.230).
Electrographic PTZ-induced seizures for 60 min after PTZ administration. The 2-test revealed no differences in the percentage of animals with PTZ-induced seizures between the Sham-NaCl 30 min (30%) and Sham-NaCl 7 d (17%) groups (referred to together as the “Sham-NaCl group”), and consequently, the data for the two groups were combined for statistical analysis. Similarly, data obtained in the Sham-ATI 30 min (22%) and Sham-ATI 7 d (20%) groups (referred to as the “Sham-ATI group”) as well as the TBI-NaCl 30 min (58%) and TBI-NaCl 7 d (67%) groups (referred to as the “TBI- NaCl group”) were combined. The percentage of rats with PTZ-induced seizures was higher in the TBI- NaCl group (21/35, 60%) than in the Sham-NaCl (4/16, 25%; p=0.021) or Sham-ATI groups (3/14, 21%;
p=0.016)(Fig. 5E). A 9-wk treatment with ATI initiated at 30 min (15/31, 48%) or at 7 d (15/28, 54%) after TBI did not reduce the percentage of animals experiencing PTZ-induced seizures compared with
21
the TBI-NaCl group when assessed at 4 wk after discontinuation of the treatment (Fig. 5E). Also, there was no difference between the ATI treatment groups (Fig. 5E).
Cumulative duration of PTZ-induced seizures for 60 min after PTZ administration. Kruskal-Wallis analysis revealed a difference in the cumulative seizure duration between groups (p=0.012). Post hoc analysis with the Mann-Whitney U test revealed that injured rats had a longer cumulative seizure duration that was not normalized by ATI treatment initiated at either 30 min or 7 d post-TBI (Fig. 5F).
3.2.4. Neuroprotection and axonal plasticity
As favorable effects were observed in the ATI 7 d treatment arm on neuroscore, beam-walking, and seizure susceptibility, we assessed whether the improvements were associated with attenuation of the cortical lesion volume and mossy fiber sprouting. As summarized in Table 7, the TBI-induced lesion volume was ~19 mm3, corresponding to a 10% to 15% volume decrease in the ipsilateral cortex.
The magnitude of the cortical lesion volume or mossy fiber sprouting in the dentate gyrus did not differ between the TBI-NaCl 7 d and TBI-ATI 7 d groups.
3.3. SR141716A 2 min and 30 min treatment arms 3.3.1. Somatomotor and cognitive recovery
Composite neuroscore and beam-walking. Kruskal-Wallis analysis revealed differences between groups in both the neuroscore and beam-walking tests over the 9-wk follow-up (p<0.05). Post hoc analysis with the Mann-Whitney U test revealed that both the vehicle and SR141716A-treated TBI groups performed worse that the corresponding sham-injured groups in the 2-min and 30-min SR141716A treatment arms (Fig. 6). SR141716A-treated TBI groups did not perform any better than the corresponding vehicle-treated groups (Fig. 6).
Morris water-maze. Kruskal-Wallis analysis revealed a difference between groups in the SR141716A 30 min treatment arm (p<0.01)(data not shown). Both the vehicle-treated and SR141716A- treated TBI groups performed worse than their corresponding sham-injured groups (both p<0.01). Rats in the TBI-SR1417161A groups, however, performed no better than rats in the TBI-Tween 30 min group (p>0.05).
3.3.2. Development of epilepsy and seizure susceptibility
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Epilepsy was diagnosed in 13% (4/30) of injured rats treated with vehicle (TBI-Tween 2 min or TBI- Tween 30 min), which did not differ from that in TBI-ATI groups (2-test). As summarized in Table 6, SR141716A treatments given at either 2 min (18% with epilepsy) or initiated at 30 min post-TBI for 9 wk (7%) did not prevent the development of epilepsy, and there was no difference compared with ATI- treated groups (2-test). Neither acute nor chronic SR141716A treatment reduced seizure susceptibility in the PTZ-test compared with vehicle treatment (data not shown).
23 4. Discussion
We tested the hypothesis that pharmacologic neurostimulation by proconvulsants enhances somato-motor and cognitive recovery and prevents epileptogenesis after TBI. Our proof-of-concept preclinical study revealed that a proconvulsant, the α2-adrenergic antagonist ATI, enhanced somato- motor recovery and reduced seizure susceptibility after TBI. Another proconvulsant, however, the CB1 receptor antagonist SR141716A, had no favorable effects.
4.1. Methodologic issues
We conducted the largest ever preclinical proof-of-concept antiepileptogenesis study. The study design was powered to monitor the effect of treatments on motor and cognitive recovery, and on seizure susceptibility. The compounds tested two compounds which have previously been used in humans: ATI to combat neurodegeneration (Pertovaara et al., 2005) and SR141716A to combat obesity (Christopoulou and Kiortsis, 2011). The drug doses and treatment time windows were selected based on previous favorable experimental results in stroke, SE and TBI models (Pitkänen et al., 2004;
Echegoyen et al., 2009). We expanded the previous studies on SR141716A by investigating both acute and delayed treatment time windows, but demonstrated no benefit.
The relatively high mortality in the ATI treatment arms was apparently related to the repeated anesthesia needed to replace the Alzet minipumps used to deliver ATI or vehicle every 4 wk. As SR141716A has been used as an anti-obesity drug, we expected to see weight loss in the treated animals. Recovery of the post-injury weight loss, however, was comparable between all treatment groups. Also, none of the treatments affected rectal temperature, excluding the possibility that a change in the body temperature influenced the study outcome. Finally, despite the fact that both ATI and SR141716A act as proconvulsants, we did not observe enhanced frequency of spontaneous seizures in the 1st EEG recording performed during the last week of chronic treatment.
4.2. Atipamezole, but not SR141716A, improved post-TBI behavioral recovery
ATI treatment showed a substantial favorable effect on motor recovery both in the beam-walking and neuroscore tests. The effect was observed regardless of whether treatment was initiated at 30 min or 7 d post-TBI, suggesting a wide post-injury therapeutic window. In the beam-walking test, the effect was already observed at 1-wk after treatment initiation, and lasted throughout the 9-wk follow-up. In
24
the neuroscore test, however, the evolution of the favorable effect was slower, becoming significant at 1-month after treatment initiation. We assume that the slow development of the treatment effect is related to enhanced axonal plasticity rather than the mere presence of the drug in the brain. In this regard, it is interesting that α2A-adrenoceptors are located in the membrane growth cones and later in the distal segments of axons in rat pheochromocytoma cells exposed to nerve growth factor (Olli- Lähdesmäki et al., 1999). Further, induction of cyclic adenosine monophosphate formation by α2- blockade is reported to activate the cyclic adenosine monophosphate response element binding protein pathway and subsequent transcription of plasticity-associated genes (Jansson et al., 1998;
Waltereit and Weller, 2003). Finally, α2-adrenoceptors in the song systems of male European starlings show seasonal variation, suggesting their role in physiologically relevant plastic responses (Riters et al., 2002).
Our data are comparable to the findings of a previous study in a rat model of stroke induced by unilateral medial cerebral artery occlusion, in which a comparable dose of ATI improved the recovery of limb function in limb-placing test when ATI treatment was initiated at 2 d post-stroke (Barbelivien et al., 2002). Our data also are consistent with those of previous studies using vagal nerve stimulation demonstrating that increasing the cortical noradrenergic activity improved motor recovery after TBI (Roosevelt et al., 2006; Smith et al., 2005). Like after stroke (Jolkkonen et al., 2000), we observed no favorable effect of ATI on the recovery of spatial learning and memory assessed with the Morris water maze. The present study suggests that ATI treatment better targets the recovery of cortical rather than hippocampal functions after lateral fluid-percussion injury–induced TBI, for which the injury epicenter is in the auditory cortex, largely preserving the perilesional somatomotor and motor areas expressing the adrenergic receptors (Ndode-Ekane et al. (2016).
Previous studies demonstrated that SR141716A has myelination-enhancing and antioxidant effects, which could address key pathologies contributing to secondary post-TBI damage (Comelli et al., 2010;
Costa et al., 2005; Thompson et al., 2005). Moreover, it increases excitability in vitro as well as in vivo in both rats and humans (Chen et al., 2007; Citraro et al., 2013; Deshpande et al., 2007; Oliviero et al., 2012). SR141716A treatment, however, showed no favorable effects on the recovery of post-TBI motor impairments. Taken together, our findings suggest that the recovery-enhancing effects of pharmacologic neurostimulation differ between transmitter systems, favouring the α2-adrenergic system over the CB1 system.
25
4.3. Atipamezole, but not SR141716A, reduced post-TBI seizure susceptibility
PTE has been estimated to present up to 20% of all acquired epilepsy, and currently there are no treatments to prevent or alleviate post-traumatic epileptogenesis. (Pitkänen and Immonen, 2014). We previously demonstrated in a rat model of temporal lobe epilepsy induced by SE that ATI treatment initiated at 1 wk post-SE did not prevent the development of epilepsy, but the epilepsy that developed was substantially milder as the daily seizure frequency was reduced from 8.4 to 0.7 (Pitkänen et al., 2004). Our data reproduced these observations in an animal model of post-traumatic epilepsy by showing that ATI – although it did not prevent the development of epilepsy – alleviated the seizure susceptibility in the PTZ-test in several measures. In particular, ATI treatment initiated at 7 d post-TBI and continued for 9 wk normalized the time to the 1st epileptiform spike and the number of EDs. It is unclear, however, why ATI initiated at 30 min post-TBI and continued for 9 wk had no favorable effects.
To reproduce the favorable effects reported by Chen et al. (2007), Echegoyen et al. (2009), and Wang et al. (2016) in a model of complex febrile seizures in P11 rats induced by hyperthermia and in the lateral fluid-percussion injury model of TBI in adult rats, we administered SR141716 treatment as a single dose at 2 min post-injury. We demonstrated no favorable disease-modifying or antiepileptogenic effects. To expand the treatment window, in another group of animals treatment with SR141716 was initiated at 30 min post-TBI and continued for 9 wk. Still, we did not find antiepileptogenic effects in any of the measures analyzed. In the 2 min treatment arm attempting to confirm the data by Echegoyen et al. (2009), we assessed seizure susceptibility at 14 wk post-TBI, a later time-point than in the study by Echegoyen et al. (2009), which may partially account for the difference. Another possible explanation relates to differences in the test used to assess seizure susceptibility (PTZ vs. kainate) and the number of animals included in the study. As no favourable effects were found in any of the parameters assessed, no additional treatment time windows were studied.
Taken together, our data support previous observations that ATI treatment is both recovery- enhancing and antiepileptogenic.
4.4. Target expression and mechanisms of action of ATI
Our supplementary studies made an attempt to understand the mechanisms of favourable recovery-enhancing effects of atipamezole. ATI, a selective 2-adrenergic antagonist with a high 2/1
adrenoceptor selectivity ratio, does not display differential affinity for 2-adrenoceptor subtypes A, B, or C in the rodent or human brain (Haapalinna et al., 1997; Lehto et al., 2015; Virtanen et al., 1989). The
26
2-adrenergic receptors are located in adrenergic, serotonergic, and dopaminergic nerve terminals (Flügge et al., 2003; Gobert et al., 1998). As expected, our immunohistochemical staining with an antibody raised against 2A-adrenergic receptors showed immunopositive punctate staining around unstained somata in the cerebral cortex, suggesting its location in presynaptic axon terminals. In correspondence with previous electronmicroscopic and functional studies (Aoki et al., 1998;
Hutchinson et al., 2011), we also found immunopositive staining in reactive astrocytes in the lesioned regions, which was most prominent during the 1st month after post-TBI. This led us to investigate whether ATI affects the release of inflammatory cytokines, as suggested by previous studies in models of brain, lung, and kidney injury (Chi et al., 2015; Yao et al., 2015; Ren et al., 2016). Moreover, whether the alleviation of inflammation-related odema by ATI could explain some of the enhanced somato- motor recovery. Our supplementary in vitro studies in astrocyte and neuronal/microglial cultures, however, did not support these ideas. Also, ATI exhibited no neuroprotective effect in vitro or in vivo.
To further explore the possibility that the favorable effect of ATI on post-TBI recovery occurred via a neuronal α2-adrenoreceptor -mediated mechanism, we induced TBI with a controlled cortical injury in mice with non-functional α2A-adrenoceptors due to D79N point mutation, previously shown to be prone to amygdala kindling (Bolkvadze and Pitkänen, 2012; Janumpalli et al., 1998). We found that at 1 month post TBI, these mutated mice were less seizure susceptible in the PTZ test than wild-type mice, indicating that genetic inactivation of α2A receptor function affects TBI-induced hyperexcitability comparable to that of ATI. Finally, we conducted an in vivo proof-of-concept study with the α2C- adrenoceptor antagonist ORM-12471 using a similar study-design as in the present study, and found no recovery enhancing or antiepileptogenic effects. Based on these supplementary data, we conclude that the favorable effects of ATI on post-TBI recovery were (largely) mediated via neuronal α2A-adrenergic receptors.
5. Conclusions
Our present and previous data indicate that ATI reduces post-TBI seizure susceptibility and post-SE epilepsy severity. ATI also enhances motor recovery after two types of brain injury (TBI – present study;
stroke – Puurunen et al., 2001). Moreover, the recently discovered favorable α2A-adrenergic receptor blockade-mediated effects on antiamyloidogenesis and insulin secretion could provide additional benefits for the use of noradrenergic α2A-antagonists in the treatment of TBI (Gribble, 2010; Chen et al., 2014). Therefore, we consider that further studies are warranted to investigate proconvulsant 2-
27
adrenergic blockade and its downstream signaling as a therapeutic target for recovery enhancement and antiepileptogenesis in order to combat the development of co-morbidities after TBI.
Acknowledgements: We thank Mr. Jarmo Hartikainen and Mrs. Merja Lukkari for excellent technical assistance. This study was supported by CURE (Citizens United for Research in Epilepsy)(AP) and The Academy of Finland (AP). Atipamezole was a gift from Orion Pharma.
Declaration of interests: Dr. Jukka Sallinen is an employee of Orion Pharma.
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