Early Toll-like receptor 4 inhibition improves immune dysfunction in the hippocampus after hypoxic-ischemic brain damage.

Background: Toll-like receptor 4 (TLR4) is implicated in neonatal hypoxic-ischemic brain damage (HIBD), but the underlying mechanism is unclear. Hypothesis: We hypothesized that TLR4 mediates brain damage after hypoxic ischemia (HI) by inducing abnormal neuroimmune responses, including activation of immune cells and expression disorder of immune factors, while early inhibition of TLR4 can alleviate the neuroimmune dysfunction. Method: Postnatal day 7 rats were randomized into control, HI, and HI+TAK-242 (TAK-242) groups. The HIBD model was developed using the Rice-Vannucci method (the left side was the ipsilateral side of HI). TAK-242 (0.5 mg/kg) was given to rat pups in the TAK-242 group at 30 min before modeling. Immunofluorescence, immunohistochemistry, and western blotting were used to determine the TLR4 expression; the number of Iba-1+, GFAP+, CD161+, MPO+, and CD3+ cells; ICAM-1 and C3a expression; and interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-10 expression in the hippocampal CA1 region. Result: Significantly increased TLR4 expression was observed in the left hippocampus, and was alleviated by TAK-242. The significant increases in Iba-1+, MPO+, and CD161+ cells at 24 h and 7 days after HI and in GFAP+ and CD3+ T cells at 7 days after HI were also counteracted by TAK-242, but no significant differences were observed among groups at 24 h after HI. ICAM-1 expression increased 24 h after HI, while C3a expression decreased; TAK-242 also alleviated these changes. TNF-α and IL-1ß expression increased, while IL-10 expression decreased at 24 h and 7 days after HI; TAK-242 counteracted the increased TNF-α and IL-1ß expression at 24 h and the changes in IL-1ß and IL-10 at 7 days, but induced no significant differences in IL-10 expression at 24 h and TNF-α expression at 7 days. Conclusion: Early TLR4 inhibition can alleviate hippocampal immune dysfunction after neonatal HIBD.

Effects of ischemic preconditioning on PDGF-BB expression in the gerbil hippocampal CA1 region following transient cerebral ischemia.

Ischemic preconditioning (IPC) is induced by exposure to brief durations of transient ischemia, which results in ischemic tolerance to a subsequent longer or lethal period of ischemia. In the present study, the effects of IPC (2 min of transient cerebral ischemia) were examined on immunoreactivity of platelet­derived growth factor (PDGF)­BB and on neuroprotection in the gerbil hippocampal CA1 region following lethal transient cerebral ischemia (LTCI; 5 min of transient cerebral ischemia). IPC was subjected to a 2­min sublethal ischemia and a LTCI was given 5­min transient ischemia. The animals in all of the groups were given recovery times of 1, 2 and 5 days and change in PDGF­BB immunoreactivity was examined as was the neuronal damage/death in the hippocampus induced by LTCI. LTCI induced a significant loss of pyramidal neurons in the hippocampal CA1 region 5 days after LTCI, and significantly decreased PDGF­BB immunoreactivity in the CA1 pyramidal neurons from day 1 after LTCI. Conversely, IPC effectively protected the CA1 pyramidal neurons from LTCI and increased PDGF­BB immunoreactivity in the CA1 pyramidal neurons post­LTCI. In conclusion, the results demonstrated that LTCI significantly altered PDGF­BB immunoreactivity in pyramidal neurons in the hippocampal CA1 region, whereas IPC increased the immunoreactivity. These findings indicated that PDGF­BB may be associated with IPC­mediated neuroprotection.

Anti-RAGE antibody attenuates isoflurane-induced cognitive dysfunction in aged rats.

Several animal studies demonstrated that the volatile anesthetic isoflurane could influence the blood-brain barrier (BBB) integrity, which involved the cognitive impairment. Increasing evidence has also shown that the receptor for advanced glycation end-products (RAGE) played a major role in maintaining the integrity of BBB. The present study aimed to determine whether the RAGE-specific antibody protects against BBB disruption and cognitive impairment induced by isoflurane exposure in aged rats. 108 aged rats were randomly divided into four groups: (1) control group (Control); (2) 4h of 2% isoflurane exposure group (ISO); (3) RAGE antibody (20µL, 2.5µg/µL) treated+4h of 2% isoflurane exposure group (anti-RAGE+ISO); (4) RAGE antibody (20µL, 2.5µg/µL) treated group (anti-RAGE). The isoflurane anesthesia resulted in the upregulation of hippocampal RAGE expression, disruption of BBB integrity, neuroinflammation, and beta-amyloid (Aß) accumulation in aged rats. In addition, significant cognitive deficits in the Morris water maze test was also observed. The antibody pretreatment resulted in significant improvements in BBB integrity. Furthermore, the expression of RAGE and proinflammatory mediators, as well as, Aß accumulation were attenuated. Moreover, the antibody administration attenuated the isoflurane-induced cognitive impairment in aged rats. These results demonstrate that RAGE signaling is involved in BBB damage after isoflurane exposure. Thus, the RAGE antibody represents a novel therapeutic intervention to prevent isoflurane-induced cognitive impairment.

Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity.

OBJECTIVE: To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. METHODS: One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. RESULTS: Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. INTERPRETATION: Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016;80:388-400.

Differential role of CCR2 in the dynamics of microglia and perivascular macrophages during prion disease.

The expansion of the microglial population is one of the hallmarks of numerous brain disorders. The addition of circulating progenitors to the pool of brain macrophages can contribute to the progression of brain disease and needs to be precisely defined to better understand the evolution of the glial and inflammatory reactions in the brain. We have analyzed the degree of infiltration/recruitment of circulating monocytes to the microglial pool, in a prion disease model of chronic neurodegeneration. Our results indicate a minimal/absent level of CCR2-dependent recruitment of circulating monocytes, local proliferation of microglia is the main driving force maintaining the amplification of the population. A deficiency in CCR2, and thus the absence of recruitment of circulating monocytes, does not impact microglial dynamics, the inflammatory profile or the temporal behavioral course of prion disease. However, the lack of CCR2 has unexpected effects including the failure to recruit perivascular macrophages in diseased but not healthy CNS and a small reduction in microglia proliferation. These data define the composition of the CNS-resident macrophage populations in prion disease and will help to understand the dynamics of the CNS innate immune response during chronic neurodegeneration.

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

OBJECTIVE: Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury. METHODS: Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed. RESULTS: The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB). INTERPRETATION: P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.

CX3CL1-induced modulation at CA1 synapses reveals multiple mechanisms of EPSC modulation involving adenosine receptor subtypes.

We characterized the role of adenosine receptor (AR) subtypes in the modulation of glutamatergic neurotransmission by the chemokine fractalkine (CX3CL1) in mouse hippocampal CA1 neurons. CX(3)CL1 causes a reversible depression of excitatory postsynaptic current (EPSC), which is abolished by the A(3)R antagonist MRS1523, but not by A(1)R (DPCPX) or A(2A)R (SCH58261) antagonists. Consistently, CX3CL1-induced EPSC depression is absent in slices from A(3)R(-/-) but not A(1)R(-/-) or A(2A)R(-/-) mice. Further, A(3)R stimulation causes similar EPSC depression. In cultured neurons, CX3CL1-induced depression of AMPA current shows A(1)R-A(3)R pharmacology. We conclude that glutamatergic depression induced by released adenosine requires the stimulation of different ARs.