A congenital CMV infection model for follow-up studies of neurodevelopmental disorders, neuroimaging abnormalities, and treatment.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.

Mild hypothermia inhibits the Notch 3 and Notch 4 activation and seizure after stroke in the rat model.

Ischemic brain injury is an important cause for seizure. Mild hypothermia of the brain or the whole body is an effective way to remit the post-stroke seizure. Our previous study revealed an implication of Notch 1 and 2 in the post-stroke seizure. This study further investigated the involvement of Notch 3 and 4 in post-stroke seizure and the effect of mild hypothermia on these two factors. A global cerebral ischemia (GCI) model was conducted in Sprague Dawley rats. Seizure activity was evaluated by the frequency of seizure attacks, seizure severity scores, and seizure discharges. Seizures were frequently occurred in the first and the second 24 h after GCI, however active whole-body cooling (mild hypothermia) and DAPT (Notch inhibitor) injection into the hippocampus, alone or in combination, alleviated seizure activity after GCI. Immunohistochemistry and Western blot assays revealed the up-regulation of Notch intracellular domain (NICD) 3 and 4 in the cerebral cortex and hippocampus following GCI, but mild hypothermia and DAPT inhibited the up-regulation of NICD 3 and 4. NF-κB, PPARα, PPARγ, cyclin D1, Sox2 and Pax6 are associated with the pathogenesis of diverse type of seizures. GCI induced NF-κB, cyclin D1, and Pax6 activity, but suppressed PPARγ. These effects of GCI were abolished by both mild hypothermia and DAPT treatment. This indicated the implication of Notch signaling in the effects of GCI. Collectively, mild hypothermia inhibits Notch 3 and Notch 4 activation and seizure after stroke in the rat model. This study adds to the further understanding of the pathogenesis of post-stroke seizures and the protective mechanism of mild hypothermia.

mTOR is involved in stroke-induced seizures and the anti-seizure effect of mild hypothermia.

Stroke is considered an underlying etiology of the development of seizures. Stroke leads to glucose and oxygen deficiency in neurons, resulting in brain dysfunction and injury. Mild hypothermia is a therapeutic strategy to inhibit stroke­induced seizures, which may be associated with the regulation of energy metabolism of the brain. Mammalian target of rapamycin (mTOR) signaling and solute carrier family 2, facilitated glucose transporter member (GLUT)­1 are critical for energy metabolism. Furthermore, mTOR overactivation and GLUT­1 deficiency are associated with genetically acquired seizures. It has been hypothesized that mTOR and GLUT­1 may additionally be involved in seizures elicited by stroke. The present study established global cerebral ischemia (GCI) models of rats. Convulsive seizure behaviors frequently occurred during the first and the second days following GCI, which were accompanied with seizure discharge reflected in the EEG monitor. Expression of phosphor (p)­mTOR and GLUT­1 were upregulated in the cerebral cortex and hippocampus, as evidenced by immunohistochemistry and western blot analyses. Mild hypothermia and/or rapamycin (mTOR inhibitor) treatments reduced the number of epileptic attacks, seizure severity scores and seizure discharges, thereby alleviating seizures induced by GCI. Mild hypothermia and/or rapamycin treatments reduced phosphorylation levels of mTOR and the downstream effecter p70S6 in neurons, and the amount of GLUT­1 in the cytomembrane of neurons. The present study revealed that mTOR is involved in stroke­induced seizures and the anti­seizure effect of mild hypothermia. The role of GLUT­1 in stroke­elicited seizures appears to be different from the role in seizures induced by other reasons. Further studies are necessary in order to elucidate the exact function of GLUT-1 in stroke­elicited seizures.

Synergistic effect of mild hypothermia and the Notch inhibitor DAPT against post stroke seizures.

Seizure is a serious complication of stroke, indicating poor prognosis. Notch signaling is associated with neuronal activity. Inhibition of Notch signaling suppresses seizure activity induced by kainic acid. The present study investigated the effect of the Notch inhibitor, DAPT, alone or in combination with mild hypothermia, on post-stroke seizures. A global cerebral ischemia (GCI) model was performed in Sprague Dawley (SD) male rats. Seizure activity was evaluated by the frequency of seizure attacks, seizure severity scores, and seizure discharges. Without any intervention, seizures occurred intensively between 24h and 48h following GCI. Seizure activity was confirmed using EEG monitoring. The expression of Notch intracellular domains (NICD) 1 and 2 were up-regulated in the cerebral cortex and hippocampus following GCI. DAPT was injected into the hippocampus of the rats to inhibit local Notch signaling. Active whole-body cooling was performed to maintain the core temperatures of rats at 33.5°C (mild hypothermia). Mild hypothermia and DAPT synergistically inhibited NICD 1 and 2 up-regulation, and post-stroke seizures. GCI augmented excitatory synaptic neurotransmission by up-regulating glutamate receptor subunits (GluN2A, GluA1) and the cotransporter, NKCC1, but attenuated inhibitory synaptic neurotransmission by down-regulating gamma amino acid, butyric acid (GABA), and the cotransporter, KCC2. DAPT treatment normalized the homeostasis of excitatory and inhibitory synaptic neurotransmission, suggesting that aberrant activation of Notch signaling is involved in post-stroke seizures. The present study adds to the further understanding of the pathogenesis of post-stroke seizures and the improvement of the treatment provided with hypothermia.