Epileptogenesis after traumatic brain injury in Plaur-deficient mice

Epilepsy & Behavior 60 (2016) 187–196

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Epileptogenesis after traumatic brain injury in Plaur-deficient mice Tamuna Bolkvadze, Noora Puhakka, Asla Pitkänen ⁎ Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland

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Article history: Received 27 January 2016 Revised 17 April 2016 Accepted 18 April 2016 Available online xxxx Keywords: Controlled cortical impact Epilepsy Pentylenetetrazol Seizure susceptibility Traumatic brain injury Urokinase-type plasminogen activator receptor

a b s t r a c t Binding of the extracellular matrix proteinase urokinase-type plasminogen activator (uPA) to its receptor, uPAR, regulates tissue remodeling during development and after injury in different organs, including the brain. Accordingly, mutations in the Plaur gene, which encodes uPAR, have been linked to language deficits, autism, and epilepsy, both in mouse and human. Whether uPAR deficiency modulates epileptogenesis and comorbidogenesis after brain injury, however, is unknown. To address this question, we induced traumatic brain injury (TBI) by controlled cortical impact (CCI) in 10 wild-type (Wt-CCI) and 16 Plaur-deficient (uPARCCI) mice. Sham-operated mice served as controls (10 Wt-sham, 10 uPAR-sham). During the 4-month followup, the mice were neurophenotyped by assessing the somatomotor performance with the composite neuroscore test, emotional learning and memory with fear conditioning to tone and context, and epileptogenesis with videoelectroencephalography monitoring and the pentylenetetrazol (PTZ) seizure susceptibility test. At the end of the testing, the mice were perfused for histology to analyze cortical and hippocampal neurodegeneration and mossy fiber sprouting. Fourteen percent (1/7) of the mice in the Wt-CCI and 0% in the uPAR-CCI groups developed spontaneous seizures (p N 0.05; chi-square). Both the Wt-CCI and uPAR-CCI groups showed increased seizure susceptibility in the PTZ test (p b 0.05), impaired recovery of motor function (p b 0.001), and neurodegeneration in the hippocampus and cortex (p b 0.05) compared with the corresponding sham-operated controls. Motor recovery and emotional learning showed a genotype effect, being more impaired in uPAR-CCI than in Wt-CCI mice (p b 0.05). The findings of the present study indicate that uPAR deficiency does not increase susceptibility to epileptogenesis after CCI injury but has an unfavorable comorbidity-modifying effect after TBI. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Each year, ~1.5 million people in the USA and ~2.5 million in Europe suffer traumatic brain injury (TBI) [1–3]. Over 40% of injured patients develop functional impairments, including epilepsy [2]. Posttraumatic epilepsy contributes significantly to compromised functional outcome and quality of life as it affects a substantial portion of patients with TBI [4,5]. Epidemiologic studies indicate that the risk of epilepsy increases as much as 7- to 17-fold after severe TBI, 4-fold after moderate TBI, and 1.5- to 3-fold after mild TBI [6–8]. Moreover, Englander et al. [9] reported that epilepsy after TBI is associated with an almost 3-fold increase in mortality compared with TBI without epilepsy. Identification of the precipitating factors and an understanding of the molecular mechanisms underlying the development of epilepsy after TBI are urgently needed for its prevention, early diagnosis, and treatment. One approach to search for factors that increase the susceptibility to posttraumatic epileptogenesis (PTEgenesis) is to assess the molecular networks that regulate the establishment of brain excitability during

⁎ Corresponding author. Tel.: +358 50 517 2091; fax: +358 17 16 3030. E-mail address: asla.pitkanen@uef.fi (A. Pitkänen).

http://dx.doi.org/10.1016/j.yebeh.2016.04.038 1525-5050/© 2016 Elsevier Inc. All rights reserved.

development as well as during postinjury tissue recovery. The urokinase-type plasminogen activator receptor (uPAR) interactome is part of the extracellular matrix molecular network, the “matrisome”, which regulates brain plasticity during development and after brain injury [10–12]. The uPAR-interactome comprises uPAR and its ligands, including urokinase-type plasminogen activator (uPA) and sushirepeated protein X-linked 2 (SRPX2)[13]. Several components of the uPAR-interactome are upregulated in experimental models and in human epilepsy [10–12,14–22]. Several studies have also proposed a direct link between genetic modifications of the uPAR-interactome and epileptogenesis. The uPAR is encoded by the plasminogen activator, urokinase receptor (Plaur) gene. Powell et al. [14] monitored over 200 Plaur-deficient mice and observed behavioral seizures in 6% of the animals. Further studies linked the epileptogenesis in Plaur-deficient mice to abnormalities in the development of cortical and hippocampal inhibitory circuitries [14,23–26]. Roll et al. [16] reported that Y72s and N327S polymorphisms in the human SRPX2 gene are linked to rolandic epilepsy with polymicrogyria and speech dyspraxia, but a more recent study challenged this finding [27]. Finally, we demonstrated that Plaur deficiency results in a worse epilepsy phenotype and progressive brain pathology in a status epilepticus model of epileptogenesis in the rat [28].

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The uPAR-interactome may play a role in the aftermath of experimental and human TBI. In urokinase-type plasminogen activator (Plau) -deficient mice with TBI, motor recovery is impaired, but PTEgenesis is not affected [29,30]. Immunostaining of the cortical autopsy tissue of patients with TBI revealed an accumulation of uPARexpressing infiltrating granulocytes, activated microglial cells, and endothelial cells [31]. Moreover, a high plasma level of uPA together with glycocalyx degradation markers in the vascular endothelium is associated with a 3-fold increase in post-TBI mortality in humans [32]. Finally, a recent study showed that the higher the plasma levels of cleaved soluble uPAR at the time of hospital admission, the more severe the TBI and the poorer the prognosis [33]. To elucidate the functional significance of uPAR in PTEgenesis, we examined whether Plaur deficiency modulates epileptogenesis and functional recovery after TBI. Our findings indicated that Plaur deficiency does not affect PTEgenesis but has an unfavorable comorbiditymodifying effect in a mouse model of TBI. 2. Materials and methods The study design is summarized in Fig. 1.

The bone was carefully removed without disruption of the underlying dura. Forty-five minutes after pentobarbital injection, TBI was performed with a CCI device (eCCI-6.3; VCU Health System, Department of Radiology, Virginia Commonwealth University) equipped with an electrically driven metallic piston controlled by a linear velocity displacement transducer. Briefly, the mouse was positioned in a stereotaxic frame on an adjustable table, and CCI was delivered using the following stroke (∅ 3-mm flat tip) parameters: depth: 0.5 mm from dura; velocity: 5 m/s; and dwell time: 100 ms. Sham-injured animals received identical anesthesia and craniotomy and placement in the stereotaxic frame but were not exposed to CCI brain injury. After the injury, a piece of plastic was placed over the craniotomy, and the incision was sutured. 2.3. Behavioral analysis 2.3.1. Composite neuroscore Postinjury motor function was assessed in 20 Wt (10 Wt-sham, 10 Wt-CCI) and 24 uPAR (10 uPAR-sham, 14 uPAR-CCI) mice at 2 d before the CCI and at 2 d, 1 wk, 2 wk, and 3 wk postinjury. The motor function test included a forelimb flexion test (left and right separately), hindlimb flexion test (left and right separately), and an angle-board test.

2.1. Animals Adult male mice (12–14 wk old at the beginning of the experiments) were used. Plaur-deficient (uPAR −/−) mice (originally from The Jackson Laboratories, B6.129P2-Plaurtm1Jld) were a generous gift from Dr. Van der Pol (University of Amsterdam, The Netherlands). They were backcrossed to the C57BL/6 genotype (Charles River, originally JAXC57BL/6J Stock 000664 from The Jackson Laboratory) for at least eight generations. The genotypes of the mice were determined by polymerase chain reaction. Breeding was continued as Wt or uPAR −/− homozygous lines (maximum of 10 generations before backcrossing). The mice were housed in individual cages and maintained under a normal 12-h light/12-h dark cycle, with constant temperature (22 ± 1 °C) and humidity (50–60%). Water and food were available ad libitum. 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 86/609/EEC. 2.2. Induction of controlled cortical impact (CCI) injury The mice were subjected to unilateral cortical contusion using the CCI protocol according to Smith et al. [34]. Animals were anesthetized with sodium pentobarbital (60 mg/kg; single intraperitoneal [i.p.] injection) and placed in a stereotaxic frame. The skull was exposed, and a craniotomy (5 mm in diameter) was performed with a trephine over the left parietotemporal cortex between lambda and bregma. The posterior edge of the craniotomy was adjacent to the lambdoid suture.

1 wk

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Neuroscore

3 wk

4 wk

5 wk

6 wk

2.3.1.2. Angle-board test. The sensorimotor deficit was assessed as the animal's ability to stand on an inclined angle board face up, down, right, or left, using the angle-board test. To set the baseline, the angle of the rubber board was set to 50°. The mouse was placed on the board first in the vertical direction (face upwards). The angle of the board was then either increased or decreased by a 2.5° step based on the ability of the mouse to stand still (nose upwards) for 5 s on the inclined board. After setting the baseline angle, the mouse was turned downwards, then to the right horizontal direction, and finally to the

Electrode implantation

FC TBI

2.3.1.1. Forelimb and hindlimb function. To assess forelimb function, a mouse was suspended by its tail. The ability of the mouse to grasp the top of the cage when the mouse was lowered toward the cage top was scored from 4 (preinjury level) to 0 (severely impaired) as follows: Score 4, instant response and forelimbs extend forward; Score 3, forelimbs extend fully forward, but response lacks strength; Score 2, some limb spasms and forelimbs extend perpendicular or parallel to body plane; Score 1, some response as forelimbs extend mainly parallel to the body plane; and Score 0, no response. To score hindlimb function, one hindlimb of the mouse was placed on the top of the cage and then gently and quickly pulled back by its tail, observing the pattern of toe spread and hindlimb extension during the suspension, as follows: Score 4, instant response, hind limbs extend fully, feet flip back, and toes spread; Score 3, hind limbs extend back fully, but motion lacks strength; Score 2, some extension of limbs (not full), feet flip back but lack strength; Score 1, some response, but hind limbs do not extend; and Score 0, no response. Forelimb and hindlimb tests were evaluated separately for the right and left sides.

7 wk

8 wk

9 wk

10 wk

PTZ 11 wk

12 wk

13 wk

14 wk

Histology 15 wk

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vEEG

Fig. 1. Study design. Traumatic brain injury (TBI) was induced in Wt (n = 10) and Plaur-deficient (uPAR) mice (n = 16) by controlled cortical impact. Follow-up at 4 months included neuroscore, fear-conditioning (FC) test to tone and context, and continuous videoelectroencephalographic (vEEG) monitoring. Cortical electrodes were implanted into the skull at 10 wk post-CCI. The first 2-wk vEEG was started at 12 wk post-TBI. In the end of the 2nd monitoring week, mice were subjected to the pentylenetetrazol (PTZ) test to assess seizure susceptibility. Then, vEEG monitoring was continued for another 1 wk. At the end of the monitoring period, the mice were perfused for histology.

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left horizontal direction, and the highest angle for each location was recorded. The angle board score was calculated as the difference from the preinjury performance as follows: 4, 0° — no difference in the angle; 3, less than 2.5° from the baseline; 2, less than 5° from the baseline; 1, less than 7.5° from the baseline; and 0, less than 10° from the baseline. The maximum composite neuroscore was 20. In addition, two other parameters were calculated: Δi — motor impairment; i.e., the difference between the composite neuroscore at baseline and at 2 d post-TBI; Δ r2 wk — motor recovery, i.e., the difference between the composite neuroscore assessed at 2 d and at 14 d post-TBI; and Δr3 wk — motor recovery, i.e., the difference between the composite neuroscore assessed at 2 d and at 21 d post-TBI.

CCI) were placed in Plexiglas cages (size: 30 × 18 × 21 cm) where they could move freely (one mouse/cage) and connected to the recording system with commutators (SL6C, Plastics One Inc.). The EEG was recorded using the Nervus EEG recording system connected to a Nervus magnus 32/8 amplifier (Taugagreining, Iceland) and filtered (high-pass filter cutoff: 0.3 Hz; low-pass filter cutoff: 100 Hz). The behavior of the animals was taped using a WV-BP330/GE video camera (Panasonic, Japan) that was positioned in front of the cages and connected to an SVT-N72P time lapse VCR (Sony, Japan) and a PVM145E video monitor (Sony, Japan). A wide-angle lens permitted simultaneous videotaping of up to eight animals. Type WFL-II/LED15W infrared light (Videor Technical, GmbH, Germany) was used at night to allow for continuous 24-h/d video monitoring.

2.3.2. Fear-conditioning test Emotional learning was tested in Wt (10 Wt-sham, 8 Wt-CCI) or uPAR (10 Wt-sham, 11 Wt-CCI) groups at 6 wk post-CCI using the fear-conditioning task to tone (conditioned stimulus, CS) paired with footshock (unconditioned stimulus, US) as described previously [35]. Briefly, the test was performed over 4 successive days. On day 0, the mice were habituated in the fear-conditioning box (Coulbourn Instruments Inc., Allentown, PA, USA) for 20 min with lights on. On days 1 and 2, a tone (20 s, 10 kHz, and 75 dB) was coterminated with an electric footshock (0.5 s, 0.5 mA; Coulbourn Instruments Inc.). Footshock was delivered during the last 500 ms of the tone. The combination of tone and footshock was repeated twice, and the time between tones varied randomly from 1 to 5 min. On day 3 (testing), the mice were taken to a novel environment (a novel box in a novel room and a novel odor used for cleaning) where they were exposed to the CS stimulus (the same tone used for training). Duration of freezing was assessed by observing videotapes and using stopwatches to measure freezing time. Freezing time was defined as the absence of all movement except respiratory-related movements. On testing days 1–3, freezing during the pre-CS period [20-s period immediately preceding the onset of the CS (tone)] was used as a measure of contextual fear conditioning, and freezing during the 20-s delivery of the CS was used to measure cued fear conditioning. All behavioral tests were performed in a soundproof room with a familiar environment in a blinded manner. Each test was carried out during the light phase of the light/dark cycle.

2.3.3.3. Analysis of video-EEG. Digital EEG files were analyzed manually by browsing the entire file on the computer screen. An electroencephalographic seizure was defined as a high-amplitude rhythmic discharge that clearly represented an atypical EEG pattern (repetitive spikes, spike-and-wave discharges, and slow waves) and lasted N5 s. An electrographic epileptiform discharge was defined as a rhythmic transient (≥ 1 s but b5 s) containing spikes and uniform sharp waves.

2.3.3. Video-EEG monitoring of spontaneous epileptiform activity 2.3.3.1. Electrode implantation. Electrodes were implanted at 10 wk postCCI, as described previously by Bolkvadze and Pitkänen [36]. Briefly, the mice (9 Wt-sham, 8 Wt-CCI, 9 uPAR-sham, 10 uPAR-CCI) were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and inserted into a stereotaxic frame, and a stainless steel screw electrode (1 mm in diameter) was placed ipsilaterally just rostralateral to the midline of the craniotomy. Another recording electrode was positioned contralaterally to the region corresponding to the center of the craniotomy (Fig. 4D). A reference electrode was inserted into the skull above the right frontal cortex. A ground electrode was inserted into the occipital bone over the cerebellum. All electrodes were connected to a plastic pedestal (MS 363 Plastics One Inc., Roanoke, VA), which was cemented onto the skull with dental acrylic. The animals were then allowed to recover for at least 1 wk before starting the recordings. If a headset was lost during the experiment, it was reimplanted (no more than once), and monitoring was continued as scheduled. Note that electrode implantation procedures and video-EEG monitoring protocols were similar in both models. 2.3.3.2. Video-EEG monitoring. Long-term (24/7) video-EEG monitoring was started at 12 wk post-TBI, as previously described by Nissinen et al. [37], with adaptation of the rat protocol for mice. Briefly, Wt mice (9 Wt-sham, 8 Wt-CCI) or uPAR mice (9 uPAR-sham, 10 uPAR-

2.3.3.4. Analysis of behavioral seizures. When an electrographic seizure was detected, the behavioral severity was analyzed from the matching video recording. Behavioral seizure activity was scored according to a slightly modified Racine's scale [38]. 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: biforelimb clonus Score 4: forelimb clonus and rearing Score 5: forelimb clonus with rearing and falling. 2.3.4. Assessment of seizure susceptibility with pentylenetetrazol (PTZ) test The PTZ test was performed after the first 2-wk video-EEG monitoring at 14 wk post-CCI to determine whether the genotype affected postTBI seizure susceptibility (Fig. 1; 8 Wt-sham, 7 Wt-CCI, 8 uPAR-sham, 9 uPAR-CCI). We administered a subconvulsant dose (50 mg/kg, i.p.) of PTZ (1,5-pentamethylenetetrazole, 98%, Sigma-Aldrich YA-Kemia Oy, Finland) that was dissolved in sterile 0.9% saline [36]. Each mouse received a single injection of PTZ. The outcome measures were latency to the first spike, total number of spikes, latency to the first electrographic seizure, occurrence of seizure (% of mice with seizure), and mortality (%) during the 60 min after PTZ administration. Spikes were defined as high-amplitude (twice baseline) sharply contoured waveforms with a duration of 20–70 ms. For statistical analysis of the number of spikes, we included only those animals that survived for at least 60 min following the PTZ injection. The total number of spikes did not include the electrographic seizure events. After the 60-min period, the animals remained under continuous video-EEG monitoring for another week. 2.4. Histology 2.4.1. Fixation After completing the last video-EEG monitoring at 16 wk post-CCI (Fig. 1), animals (7 Wt-sham, 6 Wt-CCI, 7 uPAR-sham, 7 uPAR-CCI) were anesthetized and perfused according to the Timm fixation protocol [39]. Briefly, mice were perfused with 0.37% sulfide solution (5 ml/min, 4 °C) for 10 min followed by 4% paraformaldehyde in 0.1-M sodium phosphate buffer, pH 7.4 (5 ml/min, 4 °C) for 10 min. The brains were removed from the skull and postfixed in buffered 4% paraformaldehyde for 4 h (at 4 °C) and then cryoprotected in a solution containing 20% glycerol in 0.02-M potassium phosphate buffered saline, for 24 h. The brains were blocked, frozen on dry ice, and stored at − 70 °C until cut. The brains were sectioned in the coronal plane (25 μm, 1-in-5 series) with a sliding microtome. The sections were

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stored in a cryoprotectant tissue-collecting solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium phosphate buffer) at − 20 °C until processed. 2.4.2. Nissl staining The first series of sections was mounted on glass slides (covered by 0.5% gelatin, Sigma G-2500) and stained with thionin to identify the cytoarchitectonic boundaries of different brain areas and to assess the distribution and severity of damage after TBI. 2.4.3. Cavalieri estimation of cortical volumes We used a previously described cortical index to quantify the extent of the contusion [40,41]. The reduction in cortical volume was calculated by dividing the total cortical volume of the injured hemisphere by the total cortical volume of the uninjured hemisphere. The relative size of the cortical damage was expressed as a ratio (RatioCx). Briefly, the RatioCx was derived by calculating the cortical volume on the lesioned (left) and contralateral (right) sides by systematic random sampling and Cavalieri estimation. Primarily, the areas of the injured (left) and noninjured (right) neocortex were manually outlined from thionin-stained sections throughout the brain (1-in-5 series of 25-μmthick sections; every other section was included in the analysis, i.e., the sections were 250 μm apart from each other) using the AccuStage MDPlot 5.3 graphical program and MD3 Microscope Digitizer (AccuStage, Shoreview, MN) connected to a Leica DMRB microscope. The volumes were then calculated using the Cavalieri estimation, as previously described by Kharatishvili et al. [41]: VðCxÞ¼ t  aðsÞ  ΣSðCxÞ; where t equals the section thickness (i.e., 250 μm), a(s) equals the cortical area (mm2) associated with each section, and ΣS(Cx) is the total number of sections per mouse. Histopathologic analysis was performed by an observer blinded to the EEG data of the animals.

supragranular region and the inner molecular layer; Score 3 — almost a continuous band of granules in the supragranular region and inner molecular layer; Score 4 — continuous band of granules in the supragranular region and in the inner molecular layer; and Score 5 — confluent and dense laminar band of granules that covers most of the inner molecular layer, in addition to the supragranular region. 2.5. Statistics Statistical analysis was performed using SPSS for Windows (version 19.0). Differences in the electrophysiologic data between injured and control mice were analyzed using the Mann–Whitney U test. Differences in mortality and number of mice developing epileptiform activity during the PTZ test were analyzed using the chi-square test. Correlations were assessed using the Spearman rank correlation coefficient. Nonparametric tests were used to analyze the neuroscore test results, following Kruskall–Wallis ANOVA to test the significance of the difference between the groups. If a significant difference was detected, the Mann–Whitney U test was performed as a post hoc test to assess the differences between the two groups. Wilcoxon signed-rank test was used to assess differences compared with baseline performance within each group. A general linear model (repeated measures ANOVA) was used to test the fear-conditioning data. To assess whether different animal groups separated into their own clusters, we performed unsupervised hierarchical clustering in R environment (version 3.0.1) (http//www. R-project.org/) using the gplots package. Animals were ordered in a clustering heat map with the single-linkage method together with the Manhattan distance measurement. Clusters (1–3) were identified from the dendrogram. Variables included in the analysis were r 1 wk, r 2 wk, cortex volume ratio, latency to the first spike, latency to the first seizure, and number of spikes. Animals with more than three missing values among the variables were excluded from the analysis. Data are expressed as mean ± SD. The level of significance was set at p b 0.05. 3. Results

2.4.4. Neurodegeneration severity The severity of the neuronal damage in the pyramidal cell layer of the CA1 and CA3 subfields of the hippocampus proper and in the granule cell layer and hilus of the dentate gyrus was analyzed bilaterally from thionin-stained preparations. Damage was scored according to Freund et al. [42] as follows: 0 = no cell loss, 1 = less than 20% of neurons lost, 2 = 20%–50% of neurons lost, and 3 = over 50% of neurons lost. Ten consecutive sections were analyzed blind with regard to the experimental group. The severity of neurodegeneration in each subfield from 10 successive sections (1-in-5 series, 25 μm thick) was scored, and the mean score was used for statistical analysis. 2.4.5. Timm staining and analysis of mossy fiber sprouting Mossy fiber sprouting was analyzed from an adjacent series of sections stained with Timm sulfide/silver method [39]. For staining, all coronal sections including the hippocampus were mounted on gelatincoated slides and dried at 37 °C. Staining was performed in the dark. The working solution containing gum Arabic (300 g/l), sodium citrate buffer (25.5 g/l citric acid monohydrate and 23.4 g/l sodium citrate), hydroquinone (16.9 g/l), and silver nitrate (84.5 mg/l) was poured into the staining dish. The sections were developed until an appropriate staining intensity was attained (60–75 min). The slides were then rinsed under tap water for 30 min and placed in 5% sodium thiosulfate solution for 12 min. Finally, sections were dehydrated through an ascending series of ethanol, cleared in xylene, and cover-slipped with DePeX® mounting medium. The density of mossy fiber sprouting was analyzed along the septotemporal axis of the hippocampus bilaterally [43,44]. Sprouting was semiquantitatively scored from 0 to 5: Score 0 — no granules; Score 1 — sparse granules in the supragranular region and in the inner molecular layer; Score 2 — granules evenly distributed throughout the

3.1. Mortality 3.1.1. Acute post-CCI mortality Acute injury-related mortality within the 48 h post-CCI was 0% (0/10) in the Wt-CCI group and 13% (2/16) in the uPAR-CCI group (p N 0.05; Table 1). Consequently, 10 Wt-sham, 10 Wt-CCI, 10 uPARsham, and 14 uPAR-CCI mice were tested in composite neuroscore at 2 d post-CCI. 3.1.2. Late mortality Two of 10 mice (20%) in the Wt-CCI group and 3 of 14 mice (21%) in the uPAR-CCI group died after the neuroscore (cause of death unknown). Thus, 10 Wt-sham, 8 Wt-CCI, 10 uPAR-sham, and 11 uPARCCI mice were available for the fear-conditioning test.

Table 1 Effect of genotype and injury on the mortality and occurrence of spontaneous electrographic seizures. A continuous 2-wk video-EEG was performed starting at 12 wk after controlled cortical impact (CCI) injury. At the end of the 2-wk monitoring, animals underwent pentylenetetrazol (PTZ) seizure susceptibility test. The second videoelectroencephalogram recording lasting for 1 wk was started right after the PTZ test. Animal group

Acute post-TBI mortality

Wt-sham Wt-CCI uPAR-sham uPAR-CCI

0/10 (0%) 0/10 (0%) 0/10 (0%) 2/16 (13%)

Spontaneous seizures Before PTZ test

After PTZ test

0/9 (0%) 0/8 (0%) 0/9 (0%) 0/10 (0%)

0/7 (0%) 1/6 (14%) 0/7 (0%) 0/7 (0%)

Abbreviations: TBI, traumatic brain injury; uPAR, urokinase-type plasminogen activator receptor; Wt, wild type. Percentage of animals is in parenthesis. Statistical significances: there were no differences between the animal groups in any of the parameters.

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Thereafter, 1 Wt-sham, 1 uPAR-sham, and 1 uPAR-CCI died before the electrode implantation. Consequently, the first video-EEG recording at 12 wk post-CCI was started in 9 Wt-sham (one of which died on day 5 of the EEG recording and another of which died during the PTZ test), 8 Wt-CCI (one of which died on day 6 of the EEG recording and another of which died during the PTZ test), 9 uPAR-sham (one of which died on day 8 of the EEG recording and another of which died during the PTZ test), and 10 uPAR-CCI (one of which died on day 4 of the EEG recording and another two died during the PTZ test). Therefore, the second video-EEG recording after the PTZ test was performed in 7 Wt-sham, 6 Wt-CCI, 7 uPAR-sham, and 7 uPAR-CCI mice (Table 1). Taken together, the follow-up mortality was 40% in the Wt and 38% in the uPAR groups (p N 0.05). 3.2. Behavioral analysis 3.2.1. Assessment of motor function after CCI The baseline neuroscore was similar in all groups (Fig. 2). Assessment of the injury effect at 2 d, 1 wk, and 2 wk post-CCI revealed a significant motor impairment both in the Wt-CCI and uPAR-CCI groups compared with the corresponding sham-injured groups (all p b 0.001). Moreover, we found a genotype effect in the neuroscore as the uPARCCI group had a greater motor impairment than the Wt-CCI group at 1 wk post-TBI (p b 0.05; Fig. 2). 3.2.2. Fear-conditioning test to tone and context 3.2.2.1. Conditioning to context. On days 1 and 2, uninjured animals did not show any genotype effect (Wt-sham vs. uPAR-sham, p N 0.05; Fig. 3). Also, there was no difference between the Wt-CCI and Wtsham groups (both p N 0.05). The uPAR-CCI group exhibited reduced contextual conditioning compared with the uPAR-sham group on day 2 (p b 0.05). Comparison of the two injured groups revealed a genotype effect as the uPAR-CCI group exhibited less freezing behavior than the Wt-CCI group on training days 1 and 2 (both p b 0.05). In the new environment on testing day 3, there were no differences between groups. 3.2.2.2. Conditioning to tone. In uninjured mice, the freezing time to tone on days 1 and 2 was not affected by the genotype (Wt-sham vs. uPARsham, p N 0.05; data not shown). In the Wt group, no injury effect was detected as there was no difference between the Wt-CCI and Wt-sham groups (p N 0.05). In the uPAR animals, however, there was an injury effect as the freezing was reduced in the uPAR-CCI group compared with that in the uPAR-sham group (p b 0.05). Comparison of the two injured groups on day 2 revealed a genotype effect as the freezing Wt-sham Wt-CCI uPAR-sham uPAR-CCI

20

*** ***

15

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10

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*** #

2d

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5 0 -2 d

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3 wk

Follow-up time Fig. 2. Injury and genotype effects on composite neuroscore. uPAR deficiency delays postTBI recovery of the neurologic functions as assessed by the composite neuroscore. Note that, at 2 d post-TBI, the neuroscore was similarly decreased in the Wt-CCI and uPARCCI groups, indicating an injury effect but no genotype effect. At 1 wk post-TBI, the uPAR-CCI group performed worse than the Wt-CCI-group, indicating a genotype effect on post-TBI recovery. By the end of the 3rd post-TBI week, all treatment groups performed similarly in the neuroscore test. Statistical significance: ***p b 0.001 compared with the corresponding control group (Mann–Whitney U test); #p b 0.05 compared with the Wt-CCI group (Mann–Whitney U test).

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Context Wt-sham Wt-CCI uPAR-sham uPAR-CCI

# day 1

* # day 2

test

Fig. 3. Injury and genotype effects on fear conditioning to context. Controlled cortical impact (CCI) did not affect the conditioning to context in Wt mice. We detected an injury effect in the Plaur-deficient mice as the uPAR-CCI group performed more poorly than the uPAR-sham group on day 2. Moreover, there was also a genotype effect as the uPAR-CCI group performed more poorly than the Wt-CCI group on both days 1 and 2. In the new environment on day 3, there was no difference between groups. Statistical significance: *p b 0.05 compared with the corresponding control group (Mann–Whitney U test); #p b 0.05 compared with the Wt-CCI group (Mann–Whitney U test).

time was lower in the uPAR-CCI group compared with that in the Wt-CCI group (p b 0.05). On day 3 in the new environment, freezing time did not differ between the treatment groups (p N 0.05). 3.2.3. Occurrence of spontaneous seizures The data are summarized in Table 1. None of the sham-operated mice had seizures. Only 1 of 7 (14%) mice in the Wt-CCI group had a spontaneous seizure (duration: 33 s; behavioral severity score: 4) at the end of week 3 of video-EEG monitoring, that is, 7 d after the PTZ test (Fig. 4E). None of the mice in the uPAR-CCI group had spontaneous seizures. 3.2.4. PTZ seizure susceptibility test After the first 2 wk of video-EEG monitoring (i.e., ~14 wk post-CCI), seizure susceptibility was tested by administering PTZ under video-EEG surveillance. 3.2.4.1. Mortality. Mortality after PTZ administration is summarized in Table 2. There was no difference between the Wt-CCI (1/7 mice, 17%) and Wt-sham groups (1/9 mice, 13%; p N 0.05; χ2 test). Similarly, no difference was detected between the uPAR-CCI (2/9 mice, 22%) and uPARsham groups (1/8 mice, 13%). Genotype did not affect mortality in either the sham-operated or injured mice in the PTZ test. 3.2.4.2. Latency to the first epileptiform spike. Data are summarized in Table 2. Assessment of the latency to the first epileptiform spike did not show a genotype effect (no difference between two shamoperated or injured groups). We found an injury effect, however, as the latency to the first epileptiform spike was shorter in the Wt-CCI (n = 6; 127 ± 20 s) than in Wt-sham group (n = 8; 332 ± 170 s; p b 0.05). Moreover, in 50% (3/6 mice) of the mice in the Wt-CCI group, the latency to the first epileptiform spike was shorter than 1 SD below the mean latency in the Wt-sham group. Similarly, mean latency to the first spike was shorter in the uPAR-CCI group (n = 9; 97 ± 67 s) than in the uPAR-sham group (n = 8, 265 ± 121 s; p = 0.01). In 55% (59 mice) of the mice in the uPAR-CCI group, the latency to the first spike was shorter than 1 SD below the mean in the uPAR-sham group. 3.2.4.3. Total number of spikes during the 60 min after PTZ administration. No genotype effect on the total number of spikes during the 60 min post-PTZ was detected (no difference between two injured or shaminjured groups). We found an injury effect, however, as the total number of spikes was higher in the Wt-CCI (n = 5; 122 ± 77) than in the Wt-sham group (n = 8; 49 ± 30; p b 0.05; Table 2). Also, the total number of spikes was higher in the uPAR-CCI (n = 7; 187 ± 85) than in the uPAR-sham group (n = 8; 75 ± 48; p b 0.05; Table 2).

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A

B HC

D

1.0

**

**

0.5

0

sham CCI Wt

Ground Reference

5 mm

1.5

Recording

Ratio CX

C

sham CCI uPAR

E

Fig. 4. (A) A photomicrograph of a thionin-stained coronal section from a Wt-CCI mouse that was subjected to CCI-induced TBI 4 months earlier. The mouse had a spontaneous seizure during the 3rd week of the video-EEG recording. The mice also exhibited increased seizure susceptibility in the PTZ test as the latency to the first spike was 127 s (Wt-sham 332 ± 170 s). (B) A section from a uPAR-CCI mouse with CCI-induced TBI 4 months earlier. The mouse did not show any spontaneous seizures, but it did exhibit increased seizure susceptibility in the PTZ test as the latency to the first spike was only 91 s (uPAR-sham 265 ± 121 s). In panels A and B, the open arrow indicates the CCI-induced cortical injury. (C) The mean ratio of the ipsilateral vs. contralateral cortical volume (RatioCx) (y-axis) was lower in the Wt-CCI group than in the Wt-sham group. Similarly, the mean RatioCx was reduced in the uPAR-CCI group compared with that in the uPAR-sham group. There was no genotype effect as the RatioCx did not differ between the two sham-operated or the two injured groups. (D) Schematic presentation of the dorsal surface of the mouse skull, showing the location of the craniotomy for the CCI injury and the positioning of the epidural electrodes for video-EEG monitoring. (E) A representative example of a spontaneous seizure (behavioral severity score: 4) in the Wt-CCI-injured mouse shown in panel A. Statistical significance. **p b 0.01 (compared with the corresponding control group; Mann–Whitney U test). Abbreviations: HC, hippocampus. Scale bar in panels A, B, and D equals 1 mm.

3.2.4.4. Latency to the first electrographic seizure. The data are summarized in Table 2. Assessment of latency to the first electrographic seizure induced by PTZ indicated no injury or genotype effects. 3.2.4.5. Occurrence of electrographic seizures after PTZ injection. The data are summarized in Table 2. Seizure occurrence was more common in the Wt-CCI group (5 of 7 mice, 83%) than Wt-sham group (3 of 9 mice, 33%; p b 0.05; χ2 test). Similarly, seizure occurrence was higher in the uPAR-CCI group (7 of 9 mice, 78%) than in the uPAR-sham group (5/8 mice, 63%; p N 0.05; χ2 test). There was no difference between the Wt and uPAR genotypes with sham-operation (33% vs. 66%) or with CCI (83% vs. 78%). 3.3. Histology Histologic preparations were available for analysis from 5 Wt-sham, 6 Wt-CCI, 6 uPAR-sham, and 8 uPAR-CCI mice, which were killed at 4 months after CCI.

3.3.1. Extent of cortical lesion The center of the cortical damage was −1.9 ± 0.5 mm posterior to bregma (Kruskall–Wallis, p N 0.05 between groups), and the lesion extended throughout 10 ± 5 sections (Kruskall–Wallis, p N 0.05 between the groups). In all cases, the injury extended through all layers of the cortex (Fig. 4A, B).

3.3.2. Cortical lesion volume The data are summarized in Table 3 and Fig. 4C. As expected, in the Wt-CCI group (n = 6), the mean ipsilateral cortical volume was less than that in the Wt-sham group (n = 6; 29.0 ± 4.2 mm3 vs. 43.4 ± 1.4 mm3; p b 0.01) or on the contralateral side (29.0 ± 4.2 mm3 vs. 40.0 ± 7.2 mm3; p b 0.05). Also, in the uPAR-CCI group (n = 9), the mean ipsilateral cortical volume was less than that in the uPAR-sham group (n = 6; 29.5 ± 5.6 mm3 vs. 40.0 ± 6.5 mm3; p b 0.05) or on the contralateral side (29.0 ± 5.6 mm3 vs. 38.0 ± 3.2 mm3; p b 0.01). The mean RatioCx (left/right cortical volume) was less in the Wt-CCI

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Animal Group Color Key Heatmap color Key Min

uPAR-CCI uPAR-sham Wt-CCI Wt-sham

Max

Cluster 1 Cluster 2

Cluster 3 Δ r 2wk Δ r 3wk Cortex volume Latency to the first spike Latency to the first seizure Number of spikes

Fig. 5. Unsupervised hierarchical clustering of different animal groups (Wt-sham, Wt-CCI, uPAR-sham, uPAR-CCI) based on parameters assessed in the behavioral tests and pentylenetetrazol (PTZ) test. Analysis differentiated mice into three main clusters. Note that Cluster 1 contains both Wt and uPAR sham animals, whereas Clusters 2 and 3 comprised mainly Wt-CCI and uPAR-CCI mice, respectively. Each column represents an individual animal. Each row shows the variables assessed in a given animal: recovery of motor function (Δr2 wk and Δr3 wk), volume of the remaining cortex, latency to the first spike, latency to the first seizure, and number of spikes in the PTZ test. Colors in the heat map represent variable values as a Z-score: Higher — red; lower — blue. Green color in the bar above the heat map refers to uPAR-CCI, purple to uPAR-sham, black to Wt-CCI, and dark blue to Wtsham mice. Abbreviations: CCI, controlled cortical impact; Max, maximum Z-score; Min, minimum Z-score; uPAR, urokinase type plasminogen activator receptor; Wt, wild-type. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(n = 6) than in Wt-sham group (n = 6; 0.60 ± 0.2 vs. 0.90 ± 0.05; p b 0.01). The mean RatioCx was less in the uPAR-CCI (n = 9) than in the uPAR-sham group (n = 6; 0.70 ± 0.10 vs. 1.0 ± 0.06; p b 0.01). We detected no genotype effect on the severity of cortical damage. 3.3.3. Hippocampal neurodegeneration Data from injured mice are summarized in Table 4. Both Wt and uPAR-injured groups showed only ipsilateral hippocampal neurodegeneration. There was no genotype effect on neurodegeneration. 3.3.4. Mossy fiber sprouting The data are summarized in Table 5. The mossy fiber sprouting was mild in injured groups and not affected by genotype. 3.4. Correlation between the neuroscore, cortical injury, and markers of hyperexcitability in PTZ seizure susceptibility test 3.4.1. Neuroscore We found that the more severe the cortical injury (smaller the volume), the lower the neuroscore in the Wt-CCI (n = 12; r = 0.90; p b 0.001) and uPAR-CCI groups (n = 14; r = 0.70; p b 0.01). Also, in the PTZ seizure susceptibility test, the higher the total number of spikes, the lower the neuroscore in the Wt-CCI (n = 14; r = −0.55; p b 0.05) and uPAR-CCI (n = 13; r = −0.52; p N 0.05) groups. Furthermore, the shorter the latency to the first epileptiform spike, the lower the neuroscore in the Wt-CCI (n = 17; r = 0.67; p b 0.05) and uPAR-CCI (n = 15; r = 0.68; p b 0.01) groups. 3.4.2. Cortical volume In the uPAR-CCI group (n = 17), the more severe the cortical injury (RatioCx), the higher the number of epileptiform spikes in the PTZ test

(r = −0.58; p b 0.05). No correlations were detected between the severity of cortical injury and the other parameters (latency to the first spike, latency to the first seizure) assessed in the PTZ seizure susceptibility test. Moreover, in the uPAR-CCI group, the greater the cortical injury, the shorter the freezing time in fear conditioning to context on day 3 (n = 14; r = 0.86; p b 0.001). 4. Discussion In the present study, we examined whether Plaur, a geneencoding uPAR, deficiency worsens the structural and functional outcome after TBI, particularly with regard to epileptogenesis. We induced TBI with CCI in uPAR-deficient and Wt mice. During the 4month follow-up, the mice underwent behavioral testing and assessment of epileptogenesis. Unexpectedly, uPAR deficiency did not worsen PTEgenesis or cortical/hippocampal pathology compared with wild-type mice. Recovery of the impaired somatomotor function and the learning of fear conditioning to tone and context, however, were delayed in injured Plaur-deficient mice than in injured Wt mice, indicating that uPAR deficiency induces comorbidity-modifying effects as revealed also by hierarchial cluctering (Fig. 5). 4.1. uPAR deficiency did not affect epileptogenesis after TBI Fourteen percent of the CCI-injured Wt mice were diagnosed with PTE based on the 3-wk continuous video-EEG monitoring performed during the 4th month after TBI. This is consistent with our previous observations in three independent cohorts of CCI-injured mice, in which ~ 10% of B6 C57BL/6JOlaHsd or C57BL/6J mice develop epilepsy with a low seizure frequency [3645]. The prevalence of epilepsy in these backgrounds is, however, less than that in CD1

Table 2 Effect of genotype and injury on the pentylenetetrazol (PTZ) seizure susceptibility test. Animal group

Latency to the 1st epileptiform spike (sec)

Number of spikes (during 60 min)

Latency to the 1st electrographic seizure (sec)

Occurrence of electrographic seizures

Mortality

Wt-sham Wt-CCI uPAR-sham uPAR-CCI

332 ± 170 (8) 127 ± 20 (7)⁎ 265 ± 121 (8) 97 ± 67 (9)⁎

49 ± 30 (7) 122 ± 77 (6) 75 ± 48 (7) 187 ± 85 (7)

106 ± 175 (9) 186 ± 94 (6) 672 ± 893 (8) 219 ± 262 (9)

3/8 (33%) 5/7 (83%)¤ 5/8 (63%) 7/9 (78%)¤

1/8 (11%) 1/7 (17%) 1/8 (13%) 2/9 (22%)

Abbreviations: CCI, controlled cortical impact injury; PTZ, pentylenetetrazol; uPAR, urokinase-type plasminogen activator receptor; Wt, wild type. Percentage of animals is in parenthesis. Statistical significances: ⁎p b 0.05 (Mann–Whitney U test compared with the corresponding sham-operated group); ¤p b 0.05 (χ2 test compared with the corresponding sham-operated group).

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4.2. uPAR deficiency impairs somatomotor recovery after TBI

Table 3 Effect of genotype and controled cortical impact (CCI) injury on cortical damage. Cortical volume (mm3)

Animal group

Wt-sham Wt-CCI uPAR-sham uPAR-CCI

Ipsilateral

Contralateral

43.4 ± 1.4 (5) 29.5 ± 4.21 (6)# 40.3 ± 6.5 (6) 29.0 ± 5.6 (9)⁎

45.2 ± 2.5 (4) 40.0 ± 7.2 (6) 45.3 ± 2.5 (6) 38.0 ± 3.2 (9)

Abbreviations: uPAR, urokinase-type plasminogen activator receptor; Wt, wild type. Percentage of animals is in parenthesis. Statistical significances: ⁎,#p b 0.05 (compared with the corresponding sham group; Mann–Whitney U test).

mouse, in which ~ 40% develop epilepsy with a 4-month follow-up after CCI, suggesting that genetic background affects post-TBI epileptogenesis [46,47]. We recently demonstrated a remarkable increase in PTEgenesis from 44% to 88% in an APP/PS1 mouse model of familial Alzheimer's disease, demonstrating that genomic mutations can remarkably contribute to epilepsy after CCI injury [45]. As previous studies demonstrated spontaneous seizures in 6% of naïve uPAR mice [14], it was somewhat unexpected that none of the naïve or even CCI-injured uPAR mice showed spontaneous electrographic seizures during the 3-wk continuous video-EEG monitoring. Also, contrary to previous findings [14,48,49], naïve uPAR mice did not exhibit an increased susceptibility to PTZ compared with Wt animals. Although CCI increased seizure susceptibility to PTZ in Plaur-deficient mice, the increase was not any greater than that in the Wt mice. Accordingly, PTZ administration did not increase testrelated mortality in uninjured or injured Plaur-deficient mice compared with that in Wt mice. These differences in epileptogenesis phenotypes between laboratories may relate to differences in the Plaur mutation. Our Plaur-deficient mice were generated by deleting the genomic sequence comprising exon 3 of the uPAR gene [50]. Powell et al. [14] investigated uPAR-deficient mice that were generated by deleting exons 2 through 5 of the uPAR gene. Moreover, Eagleson et al. [23] reported that the extent of interneuron loss is more severe in a mixed genetic background compared with that in a congenic C57BL/6 background. It would also be important to test whether the use of convulsants other than PTZ would reveal differences in seizure susceptibility between the genotypes. These studies highlight the dependence of the epilepsy phenotype in both naïve and injured animals on the genetic makeup of the uPAR-deficient mice, including both the type of mutation in the Plaur gene and the genetic background of the mouse carrying the gene.

The CCI induced comparable impairment in the composite neuroscore test in Wt and uPAR-deficient mice at 2 d post-TBI. Recovery in the neuroscore during the first post-TBI week, however, occurred more slowly in the uPAR-deficient mice than in the Wt mice. Wu et al. [51] previously demonstrated that sensorimotor impairment is more severe after ischemic stroke in uPAR-deficient mice compared with that in Wt mice. Moreover, treatment with recombinant uPA improved the poststroke recovery of both neurologic function and associated dendritic protrusions in Wt and uPA-deficient mice but not in uPARdeficient mice, providing support to the idea that uPAR is needed to mediate the neuroprotective effects of either endogenously or exogenously administered uPA [51]. These data highlight the potential of modulation of post-TBI recovery via uPAR-interactome. To which extent this occurs via uPAR-orchestrated intracellular signaling through its plasma membrane receptor copartners or via extracellular plasminogen-activationinduced proteolytic activity initiated by uPAR–uPA binding remains to be explored. It is important to note that tissue plasminogen activator (tPA), another extracellular proteinase-activating plasminogen, enhances neuroplasticity and improves post-TBI recovery, highlighting the potential of modulating extracellular proteolysis in tissue recovery [52].

4.3. uPAR deficiency impairs emotional learning after TBI Conditioning to the tone and context occurred similarly in shamoperated Wt and uPAR-deficient mice, indicating no genotype effect on learning of the task. Interestingly, Bissonette et al. [25] reported that uPAR-deficient mice are impaired in extinction of cued fear conditioning compared with Wt mice, which likely relates to the reduced number of parvalbumin-containing inhibitory neurons in the medial frontal cortex. Further studies are needed to assess whether the genotype effect is more prominent in the extinction of the fear-conditioning task than in the learning of the fear-conditioning task. We next assessed whether TBI impaired emotional learning and memory. We found no difference in emotional learning between the sham-operated and injured Wt mice. Similarly, Sierra-Mercado et al. [53] reported no effect of CCI injury on mouse emotional learning and memory. This probably relates to the lack of injury to key brain areas involved in the performance of the task, including the amygdala, medial frontal cortex, and hippocampus [54–56]. In uPAR-CCI mice, however, conditioning to the tone and to the context occurred more slowly than in the uPAR-sham group or Wt-CCI group, suggesting both an injury

Table 4 Severity of neurodegeneration in the ipsilateral and contralateral hippocampus and the dentate gyrus in Wt and uPAR deficient mice after CCI injury. Number of mice with damage score Ipsilateral

Contralateral

0 1 2 3 Median score p 0 1 2 3 Median score p

CA1

CA3a

CA3b

CA3c

Granule cell layer

Hilus

Wt-CCI

uPAR-CCI

Wt-CCI

uPAR-CCI

Wt-CCI

uPAR-CCI

Wt-CCI

uPAR-CCI

Wt-CCI

uPAR-CCI

Wt-CCI

uPAR-CCI

1 (20%) 1 (20%) 3 (60%) 0 (0%) 2 ns 5(100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

2 (25%) 3 (37.5%) 3 (37.5%) 0 (0%) 1 ns 8 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

1 (20%) 2 (40%) 2 (40%) 0 (0%) 1 ns 5 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

2 (25%) 2 (25%) 4 (50%) 0 (0%) 2 ns 8 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

1 (20%) 2 (40%) 2 (40%) 0 (0%) 1 ns 5 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

2 (25%) 2 (25%) 4 (50%) 0 (0%) 2 ns 8 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

1 (20%) 1 (20%) 3 (60%) 0 (0%) 2 ns 5 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

2 (25%) 2 (25%) 4 (50%) 0 (0%) 2 ns 8 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

2 (40%) 3 (60%) 0 (0%) 0 (0%) 1 ns 5 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

2 (25%) 6 (75%) 0 (0%) 0 (0%) 1 ns 8 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

3 (60%) 2 (40%) 0 (0%) 0 (0%) 1 ns 5 (100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

5 (63%) 3 (37%) 0 (0%) 0 (0%) 1 ns 8(100%) 0 (0%) 0 (0%) 0 (0%) 0 ns

Abbreviations: CCI, controlled cortical impact injury; ns, not significant; p, statistical significance; uPAR, urokinase-type plasminogen activator receptor; Wt, wild type. Numbers refer to animals with different scores (percentage in parenthesis). Ten consecutive sections were analyzed (1-in-5 series, 25 μm thick), and the damage was scored as follows: 0 = no damage, 1 = less than 20% of neurons lost, 2 = less than 20–50% of neurons lost, and 3 = over 50% of neurons lost [42]. There were no differences between the Wt-CCI (n = 5) and uPAR-CCI (n = 8) groups (χ2 test).

Abbreviations: ns, not significant; p, statistical significance; uPAR, urokinase-type plasminogen activator receptor; Wt, wild type. Numbers refer to animals with different sprouting scores (number/percentage of animals is in parenthesis). Density of sprouting was scored from 0 (no sprouting) to 5 according to Cavazos et al. [43]. The score was derived by averaging the sprouting score in 10 successive sections (1-in-5 series, 25 μm thick).

ns ns ns ns 0 0 0 0 0 (0%) 0 (0%) 0 (0%) 0 (0%) 6 (100%) 6 (100%) 6 (100%) 6 (100%) 1 (14%) 1 (17%) 1 (17%) 2 (33%) ns ns ns ns 0 (0%) 0 (0%) 0 (0%) 0 (0%) ns ns ns ns 0 1 0 1 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (33%) 1 (17%) 3 (50%) 6 (100%) 4 (67%) 5 (83%) 3 (50%) Wt-sham (6) Wt-CCI (6) uPAR-sham (6) uPAR-CCI (6)

Median 2 1

6 (100%) 6 (100%) 6 (100%) 6 (100%)

0 (0%) 0 (0%) 0 (0%) 0 (0%)

0 0 0 0

6 (86%) 5 (83%) 5 (87%) 4 (67%)

0 (0%) 0 (0%) 0 (0%) 0 (0%)

0 0 0 1

ns ns ns ns

0 (0%) 0 (0%) 0 (0%) 0 (0%)

Median 2 1 0 p 1

Ipsilateral score

0 p Median 2 1 0 0

p

Contralateral score Ipsilateral score

Septal end

Table 5 Density of mossy fiber sprouting in the dentate gyrus at 4 months after controlled cortical impact (CCI) injury.

Temporal end

2

Median

Contralateral score

p

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and genotype effect in this task. Even the injured uPAR-deficient mice, however, eventually learned the task. 4.4. uPAR deficiency did not affect the severity of the cortical or hippocampal neurodegeneration after TBI The present analysis revealed no differences in the extent or severity of the cortical or hippocampal lesions between the injured Wt and uPAR-deficient mice. Nagai et al. [57] demonstrated comparable lesion severity in Wt and uPAR-deficient mice after photothrombotic cortical stroke. These studies in TBI and stroke models, however, differ from our previous analysis in the status epilepticus model, demonstrating enhanced hippocampal neurodegeneration in uPAR-deficient mice [28]. Whether the neuroprotective effect of uPAR on secondary damage after brain injury is brain area- and/or model-specific remains to be further explored. 5. Conclusion The present study evaluated the effect of uPAR deficiency on epileptogenesis and functional recovery after TBI. Our findings indicate that uPAR deficiency does not modify post-TBI epileptogenesis. The presence of uPAR deficiency does, however, have a comorbiditymodifying effect, as the recovery of somatomotor function as well as conditioning to tone and contextual stimuli after TBI was delayed in mice with uPAR deficiency. Acknowledgments This study was supported by the Academy of Finland, COST Action ECMNET [BM1001], ERA-NET Neuron [TBI Epilepsy]. We thank Mr. Jarmo Hartikainen and Mrs. Merja Lukkari for their excellent technical assistance. Conflicts of interest The authors confirm that they have no conflicts of interests. References [1] Peeters W, van den Brande R, Polinder S, Brazinova A, Steyerberg EW, Lingsma HF, et al. Epidemiology of traumatic brain injury in Europe. Acta Neurochir 2015;157: 1683–96. [2] Corrigan JD, Selassie AW, Orman JAL. The epidemiology of traumatic brain injury. J Head Trauma Rehabil 2010;25:72–80. [3] Tagliaferri F, Compagnone C, Korsic M, Servadei F, Kraus J. A systematic review of brain injury epidemiology in Europe. Acta Neurochir 2006;148:255–68. [4] Asikainen I, Kaste M, Sarna S. Early and late posttraumatic seizures in traumatic brain injury rehabilitation patients: brain injury factors causing late seizures and influence of seizures on long-term outcome. Epilepsia 1999;40:584–9. [5] Andelic N, Hammergren N, Bautz-Holter E, Sveen U, Brunborg C, Røe C. Functional outcome and health-related quality of life 10 years after moderate-to-severe traumatic brain injury. Acta Neurol Scand 2009;120:16–23. [6] Annegers JF, Hauser WA, Coan SP, Rocca WA. A population-based study of seizures after traumatic brain injuries. N Engl J Med 1998;338:20–4. [7] Christensen J. Traumatic brain injury: risks of epilepsy and implications for medicolegal assessment. Epilepsia 2012;53(Suppl. 4):43–7. [8] Webb TS, Whitehead CR, Wells TS, Gore RK, Otte CN. Neurologically-related sequelae associated with mild traumatic brain injury. Brain Inj 2015;29:430–7. [9] Englander J, Bushnik T, Wright JM, Jamison L, Duong TT. Mortality in late posttraumatic seizures. J Neurotrauma 2009;26:1471–7. [10] Quirico-Santos T, Nascimento Mello A, Casimiro Gomes A, de Carvalho LP, de Souza JM, Alves-Leon S. Increased metalloprotease activity in the epileptogenic lesion—lobectomy reduces metalloprotease activity and urokinase-type uPAR circulating levels. Brain Res 2013;1538:172–81. [11] Liu B, Zhang B, Wang T, Liang Q-C, Jing X-R, Zheng J, et al. Increased expression of urokinase-type plasminogen activator receptor in the frontal cortex of patients with intractable frontal lobe epilepsy. J Neurosci Res 2010;88:2747–54. [12] Iyer AM, Zurolo E, Boer K, Baayen JC, Giangaspero F, Arcella A, et al. Tissue plasminogen activator and urokinase plasminogen activator in human epileptogenic pathologies. Neuroscience 2010;167:929–45. [13] Preissner KT, Kanse SM, May AE. Urokinase receptor: a molecular organizer in cellular communication. Curr Opin Cell Biol 2000;12:621–8.

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