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Birth asphyxia (BA) and subsequent hypoxic-ischemic encephalopathy (HIE) is one of the most serious birth complications affecting full-term infants and can result in severe disabilities including mental retardation, cerebral palsy, and epilepsy. Animal models of BA and HIE are important to characterize the functional and behavioral correlates of injury, explore cellular and molecular mechanisms, and assess the potential of novel therapeutic strategies. Here we used a non-invasive, physiologically validated rat model of BA and acute neonatal seizures that mimics many features of BA and HIE in human infants to study (i) the temporal development of epilepsy with spontaneous recurrent seizures (SRS) in the weeks and months after the initial brain injury, (ii) alterations in seizure threshold and hippocampal EEG that may precede the onset of SRS, and (iii) the effect of prophylactic treatment with midazolam. For this purpose, a total of 89 rat pups underwent asphyxia or sham asphyxia at postnatal day 11 and were examined over 8-10.5 months. In vehicle-treated animals, the incidence of electroclinical SRS progressively increased from 0 % at 2.5 months to 50 % at 6.5 months, 75 % at 8.5 months, and > 80 % at 10.5 months after asphyxia. Unexpectedly, post-asphyxial rats did not differ from sham-exposed rats in seizure threshold or interictal epileptiform discharges in the EEG. Treatment with midazolam (1 mg/kg i.p.) after asphyxia, which suppressed acute symptomatic neonatal seizures in about 60 % of the rat pups, significantly reduced the incidence of SRS regardless of its effect on neonatal seizures. This antiepileptogenic effect of midazolam adds to the recently reported prophylactic effects of this drug on BA-induced neuroinflammation, brain damage, behavioral alterations, and cognitive impairment in the rat asphyxia model of HIE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ethical use of laboratory animals requires that the benefits of an experimental study are carefully weighed against potential harm to the animals. In traumatic brain injury (TBI) research, ethical concerns are especially relevant to severe TBI, after which animals may experience suffering, depending on the implementation of refinement measures such as (1) postsurgical analgesia during the initial period following TBI and (2) humane endpoints. However, despite the frequent use of rodent models such as fluid percussion injury (FPI) and controlled cortical impact (CCI) in rats or mice, there is only one recent study that applied assessment of welfare to a severe TBI model, the FPI model in rats. In the present pilot study in a CCI mouse model of posttraumatic epilepsy, we assessed animal welfare by a brain injury-specific severity scoresheet. Furthermore, nest building was used as a sensitive indicator of health and welfare in laboratory mice. Sham mice that underwent craniotomy but not CCI were used for comparison. Craniotomy and CCI were performed under anesthesia with isoflurane, followed by 3 days of postsurgical analgesia with the opioid l-methadone. Mannitol was used to prevent the head pain caused by increased intracranial pressure. Using the TBI-specific scoresheet to describe and monitor potential distress in animals, moderately increased scores were determined in CCI mice only over the first 2 days after surgery, indicating that animal suffering in this model is transitory. Similarly, significantly impaired nest building was observed at 1 but not 7 days after CCI. We conclude that with effective postsurgical analgesia and mannitol behavioral recovery is rapid in mice after CCI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Year-round control of canine flea and tick infestations requires owner compliance with recommendations for regular treatments. Compliance failures can result in increased exposure of dogs to tick-borne pathogens and resurgence of flea populations. This study investigated the year-long efficacy of fluralaner 150 mg/ml injectable suspension (BRAVECTO ® injectable), developed to remove the need for multiple owner-administered, within-year treatments., Methods: This randomized, examiner-masked, non-inferiority study enrolled household dogs at veterinary clinics in Germany, France, and Spain. Each household contained a primary dog infested with ≥ 4 ticks or ≥ 5 fleas. Additional dogs in each household received the same treatment as the primary dog, either a single injection with fluralaner (15 mg/kg) on day 0, or 12 monthly treatments with oral afoxolaner (NexGard ® ) beginning on day 0. Owners presented their dogs for tick and flea assessments at visits 2 through 10 (days 14, 28, 56, 84, 112, 224, 280, 336, 365). Primary endpoints were the percentages of primary dogs free of live ticks or fleas at visit 10. Secondary endpoints were the percentage reductions of live ticks and fleas in primary dogs. All treated dogs were observed for adverse reactions throughout the study., Results: The analyzed per-protocol population included 415 primary dogs (fluralaner 279, afoxolaner 136) from 976 treated dogs (fluralaner 653, afoxolaner 323). From visits 2 through 10, ≥ 95% of primary dogs in each group were tick-free, and ≥ 93% were flea-free. The percentage of dogs free of ticks or fleas was non-inferior (P ≤ 0.0048) in the fluralaner group compared to the afoxolaner group at visit 10 and all earlier visits. Compared to baseline, fluralaner-group tick and flea counts were reduced by > 99%; afoxolaner-group tick and flea counts by > 98% and > 97%, respectively. There were no unexpected adverse events in any treated dog in either group, nor any sign of interactions between concomitantly administered vaccines and medications., Conclusions: A single subcutaneous fluralaner injection provided a level of tick and flea control equivalent to that of 12 monthly administrations of afoxolaner. The sustained fluralaner efficacy helps maintain canine health by retaining treatment with the veterinarian and eliminating treatment-compliance failures by pet owners between veterinary visits., Competing Interests: Declarations. Ethics approval and consent to participate: This study was conducted in compliance with local and national regulations. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Neonatal seizures are common in newborn infants after birth asphyxia. They occur more frequently in male than female neonates, but it is not known whether sex also affects seizure severity or duration. Furthermore, although stress and diurnal, ultradian, circadian, or multidien cycles are known to affect epileptic seizures in adults, their potential impact on neonatal seizures is not understood. This prompted us to examine the effects of season, daytime, sex, and stress on neonatal seizures in a rat model of birth asphyxia. Seizures monitored in 176 rat pups exposed to asphyxia on 40 experimental days performed over 3 years were evaluated. All rat pups exhibited seizures when exposed to asphyxia at postnatal day 11 (P11), which in terms of cortical development corresponds to term human babies. A first examination of these data indicated a seasonal variation, with the highest seizure severity in the spring. Sex and daytime did not affect seizure characteristics. However, when rat pups were subdivided into animals that were exposed to acute (short-term) stress after asphyxia (restraint and i.p. injection of vehicle) and animals that were not exposed to this stress, the seizures in stress-exposed rats were more severe but less frequent. Acute stress induced an increase in hippocampal microglia density in sham-exposed rat pups, which may have an additive effect on microglia activation induced by asphyxia. When seasonal data were separately analyzed for stress-exposed vs. non-stress-exposed rat pups, no significant seasonal variation was observed. This study illustrates that without a detailed analysis of all factors, the data would have erroneously indicated significant seasonal variability in the severity of neonatal seizures. Instead, the study demonstrates that even mild, short-lasting postnatal stress has a profound effect on asphyxia-induced seizures, most likely by increasing the activity of the hypothalamic-pituitary-adrenal axis. It will be interesting to examine how postnatal stress affects the treatment and adverse outcomes of birth asphyxia and neonatal seizures in the rat model used here., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Birth asphyxia and the resulting hypoxic-ischemic encephalopathy (HIE) are highly associated with perinatal and neonatal death, neonatal seizures, and an adverse later-life outcome. Currently used drugs, including phenobarbital and midazolam, have limited efficacy to suppress neonatal seizures. There is a medical need to develop new therapies that not only suppress neonatal seizures but also prevent later-life consequences. We have previously shown that the loop diuretic bumetanide does not potentiate the effects of phenobarbital in a rat model of birth asphyxia. Here we compared the effects of bumetanide (0.3 or 10 mg/kg i.p.), midazolam (1 mg/kg i.p.), and a combination of bumetanide and midazolam on neonatal seizures and later-life outcomes in this model. While bumetanide at either dose was ineffective when administered alone, the higher dose of bumetanide markedly potentiated midazolam's effect on neonatal seizures. Median bumetanide brain levels (0.47-0.53 µM) obtained with the higher dose were in the range known to inhibit the Na-K-Cl-cotransporter NKCC1 but it remains to be determined whether brain NKCC1 inhibition was underlying the potentiation of midazolam. When behavioral and cognitive alterations were examined over three months after asphyxia, treatment with the bumetanide/midazolam combination, but not with bumetanide or midazolam alone, prevented impairment of learning and memory. Furthermore, the combination prevented the loss of neurons in the dentate hilus and aberrant mossy fiber sprouting in the CA3a area of the hippocampus. The molecular mechanisms that explain that bumetanide potentiates midazolam but not phenobarbital in the rat model of birth asphyxia remain to be determined., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Loop diuretics such as furosemide and bumetanide, which act by inhibiting the Na-K-2Cl cotransporter NKCC2 at the thick ascending limb of the loop of Henle, have been shown to exert anti-seizure effects. However, the exact mechanism of this effect is not known. For bumetanide, it has been suggested that inhibition of the NKCC isoform NKCC1 in the membrane of brain neurons may be involved; however, NKCC1 is expressed by virtually all cell types in the brain, which makes any specific targeting of neuronal NKCC1 by bumetanide impossible. In addition, bumetanide only poorly penetrates the brain. We have previously shown that loop diuretics azosemide and torasemide also potently inhibit NKCC1. In contrast to bumetanide and furosemide, azosemide and torasemide lack a carboxylic group, which should allow them to better penetrate through biomembranes by passive diffusion. Because of the urgent medical need to develop new treatments for neonatal seizures and their adverse outcome, we evaluated the effects of azosemide and torasemide, administered alone or in combination with phenobarbital or midazolam, in a rat model of birth asphyxia and neonatal seizures. Neither diuretic suppressed the seizures when administered alone but torasemide potentiated the anti-seizure effect of midazolam. Brain levels of torasemide were below those needed to inhibit NKCC1. In addition to suppressing seizures, the combination of torasemide and midazolam, but not midazolam alone, prevented the cognitive impairment of the post-asphyxial rats at 3 months after asphyxia. Furthermore, aberrant mossy fiber sprouting in the hippocampus was more effectively prevented by the combination. We assume that either an effect on NKCC1 at the blood-brain barrier and/or cells in the periphery or the NKCC2-mediated diuretic effect of torasemide are involved in the present findings. Our data suggest that torasemide may be a useful option for improving the treatment of neonatal seizures and their adverse outcome., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: Birth asphyxia (BA) is the most frequent cause of neonatal death as well as central nervous system (CNS) injury. BA is often associated with neonatal seizures, which only poorly respond to anti-seizure medications and may contribute to the adverse neurodevelopmental outcome. Using a non-invasive rat model of BA, we have recently reported that the potent benzodiazepine, midazolam, prevents neonatal seizures in ~50% of rat pups. In addition to its anti-seizure effect, midazolam exerts anti-inflammatory actions, which is highly relevant for therapeutic intervention following BA. The 2 major aims of the present study were to examine (1) whether midazolam reduces the adverse outcome of BA, and (2) whether this effect is different in rats that did or did not exhibit neonatal seizures after drug treatment., Methods: Behavioral and cognitive tests were performed over 14 months after asphyxia, followed by immunohistochemical analyses., Results: All vehicle-treated rats had seizures after asphyxia and developed behavioral and cognitive abnormalities, neuroinflammation in gray and white matter, neurodegeneration in the hippocampus and thalamus, and hippocampal mossy fiber sprouting in subsequent months. Administration of midazolam (1 mg/kg i.p.) directly after asphyxia prevented post-asphyctic seizures in ~50% of the rats and resulted in the prevention or decrease of neuroinflammation and the behavioral, cognitive, and neurodegenerative consequences of asphyxia. Except for neurodegeneration in the thalamus, seizures did not seem to contribute to the adverse outcome of asphyxia., Interpretation: The disease-modifying effect of midazolam identified here strongly suggests that this drug provides a valuable option for improving the treatment and outcome of BA. ANN NEUROL 2023;93:226-243., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)