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INTRODUCTION
Hypoxic-ischemic brain injury is a common cause of long-term disabilities in the pediatric population. Up to 25% of the survivors of neonatal hypoxia exhibit devastating neuropsychological handicaps, including developmental delay, cerebral palsy, epilepsy, and learning disability. The lifelong consequences and medical costs mean that neonatal hypoxia-ischemia represents one of the highest burdens on society.
Excitotoxicity has been proposed as a major cause of neuronal death induced by ischemic brain injury, and hence it has been suggested that antiepileptic drugs could exert beneficial effects on ischemic brain insults.1234 Lacosamide (LCM) is a new antiepileptic drug that has a unique dual mode of action: it enhances the slow inactivation of sodium channels and regulates the action of neurotrophins via interaction with collapsin response mediator protein-2 (CRMP-2). The neuroprotective effect of LCM has been demonstrated using in vivo animal models of status epilepticus5 and traumatic brain injury.6 We hypothesized that LCM would have a neuroprotective effect on hypoxic-ischemic brain injury in neonatal rats by assuming the same underlying excitotoxicity-related brain injury mechanism.
METHODS
Animal model
On postnatal day 7, Sprague-Dawley rats were anesthetized with isoflurane, and the right common carotid artery was isolated and coagulated (UM880, Umeco, Seoul, Korea). After recovery in an incubator at 36.5℃ for 1 h, the rats were exposed to 8% oxygen (balanced gas N2, 1 ppm H2O, Jung Ang Sanso, Seoul, Korea) for 2.5 h in a glass chamber to induce a hypoxic-ischemic brain injury. After a 30-min recovery period, the rats were transferred to the dam. In this model, blood flow in the right hemisphere decreases during hypoxic exposure and recovers within 24 h after exposure, resulting in both focal brain ischemia and reperfusion.
Animal group and LCM administration
We used an oral preparation of LCM (Vimpat®, UCB Pharma, Smyrna, GA, USA). The rats were randomly divided into three groups: pre- or postfed with LCM, fed with normal saline, and sham. LCM was administered via an orogastric tube immediately before and after hypoxia-ischemia in the pretreatment group, and immediately after and 2 h later in the posttreatment group.
Neurohistopathologica findings
Qualitative measure
On postnatal day 12, the brain was removed and evaluated for gross morphological damage. Ipsilateral liquefactive cerebral infarction was evaluated by direct visual inspection (Fig. 1). The criteria for assessing cerebral infarction included pallor (loss of staining), atrophy, and tissue loss. The total numbers of animals per group were as follows: 13 sham, 16 normal-saline-fed, and 48 LCM-prefed rats at pretreatment; and 13 sham, 16 normal-saline-fed, and 25 LCM-postfed rats at posttreatment.
Quantitative measure
After finishing the gross morphological evaluations, the brain samples were frozen at −70℃, sliced at 20 µm intervals, fixed in paraformaldehyde vapor, and stained with cresyl violet. To quantify the severity of brain injury, we measured bilateral hemispheric cross-sectional areas in coronal sections at the level of the anterior commissure and mid-dorsal hippocampus using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). The mean percentage of right-sided damage relative to the intact left side was compared between LCM-fed and control rats.
Behavioral tests
Open field test
We performed the open field activity test from week 2 to week 7 after hypoxia-ischemia. The animals were placed in a black Plexiglas box (60×60×25 cm) for 10 min, during which their locomotive behaviors were monitored and analyzed using an auto-tracking system (SmarTrack, Smartech, Madison, WI, USA). Sham (n=14), normal-saline-prefed (n=16), and LCM-prefed (n=22, 100 mg/kg/dose) 8-week-old rats were included in the open field test.
Morris water maze test
The Morris water maze test was performed at week 8 after hypoxia-ischemia. The apparatus used for this experiment was a circular stainless steel tank (155 cm in diameter and 60 cm deep) containing water (24.0–26.0℃) to a depth of 40 cm. Skimmed milk was added to make the water opaque. The extramaze cues were kept constant. Three groups of 8-week-old rats were included in the Morris water maze test: sham (n=14), normal-saline-prefed (n=16), and LCM-prefed (n=22, 100 mg/kg/dose) rats. The rats learned to escape from the water by searching for a submerged (hidden) Plexiglas platform (10 cm in diameter and 38.5 cm high) in a fixed position. To familiarize them with the experimental situation, before the actual training the rats swam for 90 s with the platform removed. During each trial, the animal was placed in the water facing the wall at one of five designated starting points. Animals that failed to find the platform within 60 s were placed onto the platform and kept there for 30 s. The time to find the platform (called the escape latency) was measured. This test was performed five times per day for 5 days.
Statistical analysis
All calculations were performed using SPSS (version 20.0, SPSS Inc., Chicago, IL, USA). The proportion of subjects with brain infarction according to the LCM dose in the pretreatment and posttreatment groups was analyzed using Fisher's exact test. The measured infarction volume and behavioral test results were analyzed using the Kruskal-Wallis test. Probability values of p<0.05 were considered significant.
RESULTS
Neuroprotective effect of LCM
LCM pretreatment
The numbers of rats in the various study groups were as follows: 13 sham, 16 normal-saline-treated, and 7, 22, 8, and 11 LCM-pretreated rats in the 50, 100, 200, and 300 mg/kg/dose groups, respectively (Table 1). The percentage of subjects with brain infarction was 25.0% (4/16) for 100 mg/kg/dose LCM, which was significantly lower than in the normal-saline-treated group (100%, 10/10) (p<0.01). The mortality rate did not differ between the 100 mg/kg/dose LCM-pretreatment group and the sham group (p=1.00). However, the mortality rate increased with the LCM dose (p=0.04 and p<0.01 for 200 and 300 mg/kg/dose, respectively).
LCM posttreatment
The mortality rate increased with the LCM dose, while the infarction rate was not affected (Table 2). All survivors had infarction for the different doses of LCM, and so no effect was observed in the posttreatment group.
Comparison of tissue damage using quantitative measures
The infarction area was quantified from cross-sectional images at the level of the anterior commissure and mid-dorsal hippocampus, and the mean percentage of right-side damage was calculated. Pretreatment with LCM at 100 mg/kg/dose (n=16) significantly decreased the infarcted areas in several brain regions including the hemisphere (10.1±3.4, mean±SEM; p=0.001), hippocampus (19.6±3.3, p=0.005), cortex (15.5±3.1, p<0.001), and striatum (14.5±2.4, p<0.001) compared to the normal-saline group (n=10): 24.1±5.4, 40.5±5.1, 38.4±3.9, and 40.2±4.6, respectively (Fig. 2).
Behavioral test
Behavioral tests were performed to test the efficacy of LCM at 100 mg/kg/dose in preventing functional impairment induced by the insult.
Open field test
To evaluate locomotor activities, the total running distance was repeatedly measured from week 2 to week 7 following injury. There was no difference observed in the total running distance among sham, and normal-saline-fed, and LCM-prefed (100 mg/kg/dose) rats, suggesting that neither hypoxia-ischemia nor LCM affected the exploratory activity (p>0.05) (Fig. 3).
Morris water maze test
The Morris water maze test was performed at week 8 to compare the learning and spatial memory between the LCM-prefed and control rats. The mean escape latency was shorter for the LCM-prefed rats than for the normal-saline-prefed rats (Fig. 4), implying better spatial memory functioning in the LCM-pretreatment group (p<0.001, p=0.002, 0.002, 0.025, and 0.001 for five consecutive trials).
DISCUSSION
In this study, pretreatment with LCM at 100 mg/kg/dose significantly decreased the infarct areas in several brain regions and improved spatial learning and memory function. However, no effect of LCM was observed in the postfed group.
Cellular and synaptic events are similar to those caused by ischemia or epilepsy. In both situations, acute membrane depolarization is caused by sodium and calcium influx via voltage-gated channels as well as ligand-gated channels.178910 Accordingly, the following antiepileptic drugs that reduce sodium influx exhibit demonstrated neuroprotective effects: phenytoin,11 fosphenytoin,12 topiramate,1314 and zonisamide.15 In addition, lamotrigine16 (which inhibits both N-type calcium channels and sodium channels) and levetiracetam17 (which modulates N-type calcium channels) were also demonstrated to be neuroprotective against brain ischemia.
While the submolecular mechanism of slow inactivation is still poorly understood, LCM–as a selective modifier of slow inactivation–has been demonstrated to have additional benefits compared to classic sodium-channel-modulating anticonvulsants. Such anticonvulsants are relatively inactive in the 6-Hz psychomotor model of treatment-resistant seizures, whereas LCM has shown full effectiveness in the same test and also in the streptozotocin model for diabetic neuropathic pain. These findings also imply that the selective enhancement of slow inactivation in the voltage-gated sodium channel and/or the interaction of LCM with CRMP-2 can result in a distinct pharmacological profile compared to classic sodium-channel-modulating anticonvulsants.1819202122
CRMP-2 plays crucial roles in neuronal polarity, axonal guidance, and axonal degeneration.23 It also has roles in several neurodegenerative diseases, such as Alzheimer's disease, epilepsy, and ischemia.24 Moreover, its expression has been clarified in several in vivo animal models.25 LCM binds to CRMP-2 to modulates its function and possibly exert a synergistic effect with the slow inactivation of sodium channels as a neuroprotective strategy.
Only the pretreatment regimens in the present study showed a neuroprotective effect on hypoxia-ischemia. LCM pretreatment at 100 mg/kg/dose decreased both the incidence and volume of brain infarction. In addition, better spatial learning and memory function was observed in pretreatment group, while no beneficial effect was observed in the posttreatment group. The early prevention of inflammation and neuronal loss may reduce the overall CNS inflammatory response to injury and reduce oxidative stress, which in turn may reduce the development of chronic, long-term inflammation and associated neuronal degeneration. The early suppression of inflammation may also prevent synaptic remodeling, thereby improving functional outcomes.
A dose-dependent neuroprotective effect of LCM as shown in our study has also been reported for mouse models of traumatic brain injury and electrical status epilepticus.56 However, those studies showed that only high-dose LCM exhibit neuroprotective effects.
While LCM has a wide safety margin and fewer fatal side effects,26 higher doses (i.e., above 200 mg/kg/dose) appear to be fatal in our animal model. The cause of death is suspected to be a combined effect of respiratory depression by the sedative effect of LCM, compromised cardiac function, and prolonged hypoxic insult. The cause of death and the real safety margin could be clarified in future studies involving older rats that are large enough to allow the application of a ventilator device or blood pressure monitor.
In conclusion, the findings of this study suggest that LCM exerts a neuroprotective effect against hypoxia-ischemia in neonatal rats. This suggests that LCM pretreatment could be used as an effective neuroprotective method for neonates under hypoxic-ischemic conditions including heart surgery. Further clinical studies are needed to confirm these preclinical observations and to define an appropriate dosing regimen.
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