Bumetanide Attenuates Cognitive Deficits and Brain Damage in Rats Subjected to Hypoxia-Ischemia at Two Time Points of the Early Postnatal Period.

Neonatal hypoxia-ischemia (HI) is one of the main causes of tissue damage, cell death, and imbalance between neuronal excitation and inhibition and synaptic loss in newborns. GABA, the major inhibitory neurotransmitter of the central nervous system (CNS) in adults, is excitatory at the onset of neurodevelopment and its action depends on the chloride (Cl-) cotransporters NKCC1 (imports Cl-) and KCC2 (exports Cl-) expression. Under basal conditions, the NKCC1/KCC2 ratio decreases over neurodevelopment. Thus, changes in this ratio caused by HI may be related to neurological disorders. The present study evaluated the effects of bumetanide (NKCC cotransporters inhibitor) on HI impairments in two neurodevelopmental periods. Male Wistar rat pups, 3 (PND3) and 11 (PND11) days old, were submitted to the Rice-Vannucci model. Animals were divided into 3 groups: SHAM, HI-SAL, and HI-BUM, considering each age. Bumetanide was administered intraperitoneally at 1, 24, 48, and 72 h after HI. NKCC1, KCC2, PSD-95, and synaptophysin proteins were analyzed after the last injection by western blot. Negative geotaxis, righting reflex, open field, object recognition test, and Morris water maze task were performed to assess neurological reflexes, locomotion, and memory function. Tissue atrophy and cell death were evaluated by histology. Bumetanide prevented neurodevelopmental delay, hyperactivity, and declarative and spatial memory deficits. Furthermore, bumetanide reversed HI-induced brain tissue damage, reduced neuronal death and controlled GABAergic tone, maintained the NKCC1/KCC2 ratio, and synaptogenesis close to normality. Thereby, bumetanide appears to play an important therapeutic role in the CNS, protecting the animals against HI damage and improving functional performance.

L-2-Hydroxyglutaric Acid Administration to Neonatal Rats Elicits Marked Neurochemical Alterations and Long-Term Neurobehavioral Disabilities Mediated by Oxidative Stress.

L-2-Hydroxyglutaric aciduria (L-2-HGA) is an inherited neurometabolic disorder caused by deficient activity of L-2-hydroxyglutarate dehydrogenase. L-2-Hydroxyglutaric acid (L-2-HG) accumulation in the brain and biological fluids is the biochemical hallmark of this disease. Patients present exclusively neurological symptoms and brain abnormalities, particularly in the cerebral cortex, basal ganglia, and cerebellum. Since the pathogenesis of this disorder is still poorly established, we investigated the short-lived effects of an intracerebroventricular injection of L-2-HG to neonatal rats on redox homeostasis in the cerebellum, which is mostly affected in this disorder. We also determined immunohistochemical landmarks of neuronal viability (NeuN), astrogliosis (S100B and GFAP), microglia activation (Iba1), and myelination (MBP and CNPase) in the cerebral cortex and striatum following L-2-HG administration. Finally, the neuromotor development and cognitive abilities were examined. L-2-HG elicited oxidative stress in the cerebellum 6 h after its injection, which was verified by increased reactive oxygen species production, lipid oxidative damage, and altered antioxidant defenses (decreased concentrations of reduced glutathione and increased glutathione peroxidase and superoxide dismutase activities). L-2-HG also decreased the content of NeuN, MBP, and CNPase, and increased S100B, GFAP, and Iba1 in the cerebral cortex and striatum at postnatal days 15 and 75, implying long-standing neuronal loss, demyelination, astrocyte reactivity, and increased inflammatory response, respectively. Finally, L-2-HG administration caused a delay in neuromotor development and a deficit of cognition in adult animals. Importantly, the antioxidant melatonin prevented L-2-HG-induced deleterious neurochemical, immunohistochemical, and behavioral effects, indicating that oxidative stress may be central to the pathogenesis of brain damage in L-2-HGA.

Effects of acrobatic training on spatial memory and astrocytic scar in CA1 subfield of hippocampus after chronic cerebral hypoperfusion in male and female rats.

Chronic cerebral hypoperfusion leads to neuronal loss in the hippocampus and spatial memory impairments. Physical exercise is known to prevent cognitive deficits in animal models; and there is evidence of sex differences in behavioral neuroprotective approaches. The aim of present study was to investigate the effects of acrobatic training in male and female rats submitted to chronic cerebral hypoperfusion. Males and females rats underwent 2VO (two-vessel occlusion) surgery and were randomly allocated into 4 groups of males and 4 groups of females, as follows: 2VO acrobatic, 2VO sedentary, Sham acrobatic and Sham sedentary. The acrobatic training started 45 days after surgery and lasted 4 weeks; animals were then submitted to object recognition and water maze testing. Brain samples were collected for histological and morphological assessment and flow cytometry. 2VO causes cognitive impairments and acrobatic training prevented spatial memory deficits assessed in the water maze, mainly for females. Morphological analysis showed that 2VO animals had less NeuN labeling and acrobatic training prevented it. Increased number of GFAP positive cells was observerd in females; moreover, males had more branched astrocytes and acrobatic training prevented the branching after 2VO. Flow cytometry showed higher mitochondrial potential in trained animals and more reactive oxygen species production in males. Acrobatic training promoted neuronal survival and improved mitochondrial function in both sexes, and influenced the glial scar in a sex-dependent manner, associated to greater cognitive benefit to females after chronic cerebral hypoperfusion.