Real-Time Brain Monitoring by Near-Infrared Spectroscopy Predicts Neurological Outcome after Cardiac Arrest and Resuscitation in Rats: A Proof of Concept Study of a Novel Prognostic Measure after Cardiac Arrest

Clinical studies have demonstrated that dynamic changes in regional cerebral oxygen saturation (rSO2) after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) have a role in predicting neurological outcomes after the return of spontaneous circulation (ROSC). Our study evaluated whether the timing of rSO2 decline shortly after CPR reflects the severity of brain injury in a rat model of CA. Rats were subjected to different durations of asphyxia to produce variable severities of brain injury, due to CA. Time from ROSC to achieving the initial minimum rSO2 was defined as Tnadir. A Tnadir cut-off of 24 min had optimal sensitivity and specificity for predicting good neurological outcomes at 72 h after ROSC (AUC, 0.88; sensitivity, 89%; specificity, 86%; p < 0.01). Immunohistochemistry at 72 h post-CA revealed that the number of Fluoro-Jade B positive degenerating neurons in the hippocampus CA1 sector were markedly higher in animals with Tnadir > 24 min than that in animals with Tnadir ≤ 24 min. There was no difference in the gene expressions of cytokines and mitochondrial fission proteins in the brain at 2 h after ROSC between rats with Tnadir > 24 min and with Tnadir ≤ 24 min. In conclusion, Tnadir can be a novel predictor of good neurological outcomes after CA/CPR.

Ischemia-Induced Cognitive Impairment Is Improved via Remyelination and Restoration of Synaptic Density in the Hippocampus after Treatment with COG-Up® in a Gerbil Model of Ischemic Stroke

Cerebrovascular disease such as ischemic stroke develops cognitive impairment due to brain tissue damage including neural loss, demyelination and decrease in synaptic density. In the present study, we developed transient ischemia in the forebrain of the gerbil and found cognitive impairment using the Barnes maze test and passive avoidance test for spatial memory and learning memory, respectively. In addition, neuronal loss/death was detected in the Cornu Ammonis 1 (CA1) region of the gerbil hippocampus after the ischemia by cresyl violet histochemistry, immunohistochemistry for neuronal nuclei and histofluorescence with Fluoro-Jade B. Furthermore, in the CA1 region following ischemia, myelin and vesicular synaptic density were significantly decreased using immunohistochemistry for myelin basic protein and vesicular glutamate transporter 1. In the gerbils, treatment with COG-up® (a combined extract of Erigeron annuus (L.) Pers. and Brassica oleracea Var.), which was rich in scutellarin and sinapic acid, after the ischemia, significantly improved ischemia-induced decline in memory function when compared with that shown in gerbils treated with vehicle after the ischemia. In the CA1 region of these gerbils, COG-up® treatment significantly promoted the remyelination visualized using immunohistochemistry myelin basic protein, increased oligodendrocytes visualized using a receptor-interacting protein, and restored the density of glutamatergic synapses visualized using double immunofluorescence for vesicular glutamate transporter 1 and microtubule-associated protein, although COG-up® treatment did not protect pyramidal cells (principal neurons) located in the CA1 region form the ischemic insult. Considering the current findings, a gerbil model of ischemic stroke apparently showed cognitive impairment accompanied by ischemic injury in the hippocampus; also, COG-up® can be employed for improving cognitive decline following ischemia-reperfusion injury in brains.

Abstract 14258: Modulation of Kynurenine Pathway to Prevent Brain Injury After Cardiac Arrest and Cardiopulmonary Resuscitation in Mice

Introduction: Kynurenine pathway (KP) is emerging as one of the potential components affecting cardiac arrest (CA) outcomes. The aim of this study is to evaluate the effects of KP inhibition through genetic deletion of the rate-limiting enzyme of the KP, indoleamine-2,3-dyoxygenase (IDO) on survival and neurological outcome after CA. Methods and Results: Sixteen adult male wild-type (WT) and IDO-deleted (IDO -/- ) mice were subjected to 8 min untreated CA followed by resuscitation. At baseline heart rate and mean arterial pressure (MAP) did not differ among groups. At the time of return of spontaneous circulation, 30 and 60 min later, MAP was higher in the IDO -/- group compared to the WT one (p=0.0005). IDO -/- mice showed higher survival compared to WT at 7 days after CA (68.5% in IDO -/- vs 37.5% in WT; log rank p=0.036). Neurological function was higher in IDO -/- than in WT mice during the 7 days following CA (p=0.0124). IDO -/- mice also showed an improved locomotor function compared to WT mice (p=0.037). Brain magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) sequences showed a reduction in fractional anisotropy in the external capsule of the corpus callosum in WT mice compared to IDO -/- mice at 7 days after resuscitation (p=0.015). We then treated additional IDO -/- mice with L-kyn 15 min before CA, to revert the IDO -/- phenotype. Brain MRI with diffusion-weighted imaging (DWI) sequences and histological analysis were performed 24h after CA in WT, IDO -/- , and IDO -/- +L-Kyn mice. Brain MRI revealed restriction of water diffusivity 24h after CA in WT mice. IDO-deletion reduced water diffusion abnormalities while the beneficial effect was reverted in the L-kyn group (p=0.01). Degenerating neurons in the frontal cortex, represented as Fluoro-Jade B positive cells, were more numerous in WT compared to IDO -/- mice; L-kyn halted this IDO deletion-induced reduction in degenerating cells (p=0.05). Conclusion: KP inhibition improves survival and neurological outcome after CA. The neuroprotective effect of IDO-deletion was associated with preservation of brain white matter microintegrity and with reduction of cerebral cytotoxic edema. Reversal of these beneficial effects by L-kyn administration in IDO -/- mice further confirm the KP role in CA outcome.

Lactoferrin Protects against Methamphetamine Toxicity by Modulating Autophagy and Mitochondrial Status

Lactoferrin (LF) was used at first as a vehicle to deliver non-soluble active compounds to the body, including the central nervous system (CNS). Nonetheless, it soon became evident that, apart from acting as a vehicle, LF itself owns active effects in the CNS. In the present study, the effects of LF are assessed both in baseline conditions, as well as to counteract methamphetamine (METH)-induced neurodegeneration by assessing cell viability, cell phenotype, mitochondrial status, and specific autophagy steps. In detail, cell integrity in baseline conditions and following METH administration was carried out by using H&E staining, Trypan blue, Fluoro Jade B, and WST-1. Western blot and immuno-fluorescence were used to assess the expression of the neurofilament marker βIII-tubulin. Mitochondria were stained using Mito Tracker Red and Green and were further detailed and quantified by using transmission electron microscopy. Autophagy markers were analyzed through immuno-fluorescence and electron microscopy. LF counteracts METH-induced degeneration. In detail, LF significantly attenuates the amount of cell loss and mitochondrial alterations produced by METH; and mitigates the dissipation of autophagy-related proteins from the autophagy compartment, which is massively induced by METH. These findings indicate a protective role of LF in the molecular mechanisms of neurodegeneration.

Shape effect of cerium oxide nanoparticles on mild traumatic brain injury

The catalytic performance and therapeutic effect of nanoparticles varies with shape. Here, we investigated and compared the therapeutic outcomes of ceria nanospheres (Ceria NSs) and ceria nanorods (Ceria NRs) in an in vivo study of mild traumatic brain injury (mTBI). In vivo TBI was induced in a mouse model of open head injury using a stereotaxic impactor. Outcomes including cytoprotective effects, cognitive function, and cerebral edema were investigated after retro-orbital injection of 11.6 mM of ceria nanoparticles. Ceria nanoparticles significantly reduced fluoro-jade B (FJB)-positive cells and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, and restored mRNA levels of superoxide dismutase 1 (SOD1) and SOD2. They also decreased the cyclooxygenase-2 (COX-2) expression compared with the untreated control group. Comparing the two nanomaterials, Ceria NRs showed less stable and high-energy (100) and (110) planes, which increased the number of active sites. The Ce3+/Ce4+ molar ratio of Ceria NRs (0.40) was greater than that of Ceria NSs (0.27). Ceria NRs (0.059 ± 0.021) appeared to exhibit better anti-inflammatory effect than Ceria NSs (0.133 ± 0.024), but the effect was statistically insignificant (p = 0.190). Ceria nanoparticles also improved cognitive impairment following mTBI compared with the control group, but the effect did not differ significantly according to the nanoshape. However, Ceria NRs (70.1 ± 0.5%) significantly decreased brain water content compared with Ceria NSs (73.7 ± 0.4%; p = 0.0015), indicating a more effective reduction in brain edema (p = 0.0015). Compared with Ceria NSs, the Ceria NRs are more effective in alleviating cerebral edema following in vivo mTBI.

Use of Both Fluoro-Jade B and Hematoxylin and Eosin to Detect Cell Death in the Juvenile Rat Brain Exposed to NMDA-Receptor Antagonists or GABA-Receptor Agonists in Safety Assessment

Administration of pediatric anesthetics with N-methyl D-aspartate (NMDA)-receptor antagonist and/or γ-aminobutyric acid (GABA) agonist activities may result in neuronal degeneration and/or neuronal cell death in neonatal rats. Evaluating pediatric drug candidates for this potential neurotoxicity is often part of overall preclinical new drug development strategy. This specialized assessment may require dosing neonatal rats at postnatal day 7 at the peak of the brain growth spurt and evaluating brain tissue 24 to 48 hours following dosing. The need to identify methods to aid in the accurate and reproducible detection of lesions associated with this type of neurotoxic profile is paramount for meeting the changing needs of neuropathology assessment and addressing emerging challenges in the neuroscience field. We document the use of Fluoro-Jade B (FJB) staining, to be used in conjunction with standard hematoxylin and eosin staining, to detect acute neurodegeneration and neuronal cell death that can be caused by some NMDA-receptor antagonists and/or GABA agonists in the neonatal rat brain. The FJB staining is simple, specific, and sensitive and can be performed on brain specimens from the same cohort of animals utilized for standard neurotoxicity assessment, thus satisfying animal welfare recommendations with no effect on achievement of scientific and regulatory goals.

The Effects of Atorvastatin on Global Cerebral Ischemia-Induced Neuronal Death

(1) Background and Purpose: Global cerebral ischemia-induced severe hypoxic brain damage is one of the main causes of mortality and long-term neurologic disability even after receiving early blood reperfusion. This study aimed to test the hypothesis that atorvastatin potentially has neuroprotective effects in global cerebral ischemia (GCI). (2) Methods: We performed two sets of experiments, analyzing acute (1-week) and chronic (4-week) treatments. For the vehicle (Veh) and statin treatments, 1 mL of 0.9% saline and 5 mg/kg of atorvastatin (ATOR) were administered orally. For histological analysis, we used the following staining protocols: Fluoro-Jade B and NeuN, 4-hydroxynonenal, CD11b and GFAP, IgG, SMI71, and vWF. Finally, we evaluated the cognitive function with a battery of behavioral tests. (3) Results: The GCI-ATOR group showed significantly reduced neuronal death, oxidative stress, inflammation, and BBB disruption compared with the GCI-Veh group. Moreover, the GCI-ATOR group showed decreased endothelial damage and VV proliferation and had significantly improved cognitive function compared with the GCI-Veh group in both models. (4) Conclusions: ATOR has neuroprotective effects and helps recover the cognitive function after GCI in rats. Therefore, administration of atorvastatin may be a therapeutic option in managing GCI after CA.

Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

Background P2X7 receptor (P2X7R) is an ATP-gated nonselective cationic channel playing important roles in a variety of physiological functions, including inflammation, and apoptotic or necrotic cell death. An extracellular domain has ten cysteine residues forming five intrasubunit disulfide bonds, which are needed for the P2X7R trafficking to the cell surface and the recognition of surface epitopes of apoptotic cells and bacteria. However, the underlying mechanisms of redox/S-nitrosylation of cysteine residues on P2X7R and its role in P2X7R-mediated post-status epilepticus (SE, a prolonged seizure activity) events remain to be answered. Methods Rats were given pilocarpine (380 mg/kg i.p.) to induce SE. Animals were intracerebroventricularly infused Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME, a NOS inhibitor) 3 days before SE, or protein disulfide isomerase (PDI) siRNA 1 day after SE using an osmotic pump. Thereafter, we performed Western blot, co-immunoprecipitation, membrane fraction, measurement of S-nitrosylated (SNO)-thiol and total thiol, Fluoro-Jade B staining, immunohistochemistry, and TUNEL staining. Results SE increased S-nitrosylation ratio of P2X7R and the PDI-P2X7R bindings, which were abolished by L-NAME and PDI knockdown. In addition, both L-NAME and PDI siRNA attenuated SE-induced microglial activation and astroglial apoptosis. L-NAME and PDI siRNA also ameliorated the increased P2X7R surface expression induced by SE. Conclusions These findings suggest that PDI-mediated redox/S-nitrosylation may facilitate the trafficking of P2X7R, which promotes microglial activation and astroglial apoptosis following SE. Therefore, our findings suggest that PDI-mediated regulations of dynamic redox status and S-nitrosylation of P2X7R may be a critical mechanism in the neuroinflammation and astroglial death following SE.

Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury

Background Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Microglial/macrophage activation and neuroinflammation are key cellular events following TBI, but the regulatory and functional mechanisms are still not well understood. Myeloid-epithelial-reproductive tyrosine kinase (Mer), a member of the Tyro-Axl-Mer (TAM) family of receptor tyrosine kinases, regulates multiple features of microglial/macrophage physiology. However, its function in regulating the innate immune response and microglial/macrophage M1/M2 polarization in TBI has not been addressed. The present study aimed to evaluate the role of Mer in regulating microglial/macrophage M1/M2 polarization and neuroinflammation following TBI. Methods The controlled cortical impact (CCI) mouse model was employed. Mer siRNA was intracerebroventricularly administered, and recombinant protein S (PS) was intravenously applied for intervention. The neurobehavioral assessments, RT-PCR, Western blot, magnetic-activated cell sorting, immunohistochemistry and confocal microscopy analysis, Nissl and Fluoro-Jade B staining, brain water content measurement, and contusion volume assessment were performed. Results Mer is upregulated and regulates microglial/macrophage M1/M2 polarization and neuroinflammation in the acute stage of TBI. Mechanistically, Mer activates the signal transducer and activator of transcription 1 (STAT1)/suppressor of cytokine signaling 1/3 (SOCS1/3) pathway. Inhibition of Mer markedly decreases microglial/macrophage M2-like polarization while increases M1-like polarization, which exacerbates the secondary brain damage and sensorimotor deficits after TBI. Recombinant PS exerts beneficial effects in TBI mice through Mer activation. Conclusions Mer is an important regulator of microglial/macrophage M1/M2 polarization and neuroinflammation, and may be considered as a potential target for therapeutic intervention in TBI.

Neuronal Death in the CNS Autonomic Control Center Comes Very Early after Cardiac Arrest and Is Not Significantly Attenuated by Prompt Hypothermic Treatment in Rats

Autonomic dysfunction in the central nervous system (CNS) can cause death after recovery from a cardiac arrest (CA). However, few studies on histopathological changes in animal models of CA have been reported. In this study, we investigated the prevalence of neuronal death and damage in various brain regions and the spinal cord at early times after asphyxial CA and we studied the relationship between the mortality rate and neuronal damage following hypothermic treatment after CA. Rats were subjected to 7–8 min of asphyxial CA, followed by resuscitation and prompt hypothermic treatment. Eight regions related to autonomic control (the cingulate cortex, hippocampus, thalamus, hypothalamus, myelencephalon, and spinal cord) were examined using cresyl violet (a marker for Nissl substance) and Fluoro-Jade B (a marker for neuronal death). The survival rate was 44.5% 1 day post-CA, 18.2% 2 days post-CA and 0% 5 days post-CA. Neuronal death started 12 h post-CA in the gigantocellular reticular nucleus and caudoventrolateral reticular nucleus in the myelencephalon and lamina VII in the cervical, thoracic, lumbar, and sacral spinal cord, of which neurons are related to autonomic lower motor neurons. In these regions, Iba-1 immunoreactivity indicating microglial activation (microgliosis) was gradually increased with time after CA. Prompt hypothermic treatment increased the survival rate at 5 days after CA with an attenuation of neuronal damages and death in the damaged regions. However, the survival rate was 0% at 12 days after CA. Taken together, our study suggests that the early damage and death of neurons related to autonomic lower motor neurons was significantly related to the high mortality rate after CA and that prompt hypothermic therapy could increase the survival rate temporarily after CA, but could not ultimately save the animal.