TLR4-Pathway-Associated Biomarkers in Subarachnoid Hemorrhage (SAH): Potential Targets for Future Anti-Inflammatory Therapies.

Subarachnoid hemorrhage is associated with severe neurological deficits for survivors. Among survivors of the initial bleeding, secondary brain injury leads to additional brain damage. Apart from cerebral vasospasm, secondary brain injury mainly results from cerebral inflammation taking place in the brain parenchyma after bleeding. The brain's innate immune system is activated, which leads to disturbances in brain homeostasis, cleavage of inflammatory cytokines and, subsequently, neuronal cell death. The toll-like receptor (TLR)4 signaling pathway has been found to play an essential role in the pathophysiology of acute brain injuries such as subarachnoid hemorrhage (SAH). TLR4 is expressed on the cell surface of microglia, which are key players in the cellular immune responses of the brain. The participants in the signaling pathway, such as TLR4-pathway-like ligands, the receptor itself, and inflammatory cytokines, can act as biomarkers, serving as clues regarding the inflammatory status after SAH. Moreover, protein complexes such as the NLRP3 inflammasome or receptors such as TREM1 frame the TLR4 pathway and are indicative of inflammation. In this review, we focus on the activity of the TLR4 pathway and its contributors, which can act as biomarkers of neuroinflammation or even offer potential new treatment targets for secondary neuronal cell death after SAH.

Microglia as target for anti-inflammatory approaches to prevent secondary brain injury after subarachnoid hemorrhage (SAH).

BACKGROUND: Microglia-driven cerebral spreading inflammation is a key contributor to secondary brain injury after SAH. Genetic depletion or deactivation of microglia has been shown to ameliorate neuronal cell death. Therefore, clinically feasible anti-inflammatory approaches counteracting microglia accumulation or activation are interesting targets for SAH treatment. Here, we tested two different methods of interference with microglia-driven cerebral inflammation in a murine SAH model: (i) inflammatory preconditioning and (ii) pharmacological deactivation. METHODS: 7T-MRI-controlled SAH was induced by endovascular perforation in four groups of C57Bl/6 mice: (i) Sham-operation, (ii) SAH naïve, (iii) SAH followed by inflammatory preconditioning (LPS intraperitoneally), and (iv) SAH followed by pharmacological microglia deactivation (colony-stimulating factor-1 receptor-antagonist PLX3397 intraperitoneally). Microglia accumulation and neuronal cell death (immuno-fluorescence), as well as activation status (RT-PCR for inflammation-associated molecules from isolated microglia) were recorded at day 4 and 14. Toll-like receptor4 (TLR4) status was analyzed using FACS. RESULTS: Following SAH, significant cerebral spreading inflammation occurred. Microglia accumulation and pro-inflammatory gene expression were accompanied by neuronal cell death with a maximum on day 14 after SAH. Inflammatory preconditioning as well as PLX3397-treatment resulted in significantly reduced microglia accumulation and activation as well as neuronal cell death. TLR4 surface expression in preconditioned animals was diminished as a sign for receptor activation and internalization. CONCLUSIONS: Microglia-driven cerebral spreading inflammation following SAH contributes to secondary brain injury. Two microglia-focused treatment strategies, (i) inflammatory preconditioning with LPS and (ii) pharmacological deactivation with PLX3397, led to significant reduction of neuronal cell death. Increased internalization of inflammation-driving TLR4 after preconditioning leaves less receptor molecules on the cell surface, providing a probable explanation for significantly reduced microglia activation. Our findings support microglia-focused treatment strategies to overcome secondary brain injury after SAH. Delayed inflammation onset provides a valuable clinical window of opportunity.

Confirmation of Amblyomma americanum (Acari: Ixodidae) as a vector for Cytauxzoon felis (Piroplasmorida: Theileriidae) to domestic cats.

Amblyomma americanum was confirmed as a competent vector in the transmission of Cytauxzoon felis to domestic cats. Infection with C. felis was produced and replicated in four domestic felines by the bite of A. americanum adults that were acquisition fed as nymphs on a domestic cat that survived cytauxzoonosis. Numerous attempts to transmit C. felis with Dermacentor variabilis at the same time were not successful. All cats upon which infected A. americanum were transmission fed exhibited disease typical of cytauxzoonosis, and the eitiologic agent's presence was confirmed. Clinical signs including fever, inappetence, depression, and lethargy were observed beginning 13 d postinfestation. Pale mucus membranes, splenomegaly, icterus, and dyspnea were also observed during the course of the disease. Rectal temperatures of the C. felis-infected principal cats fluctuated from high to subnormal before returning to the normal range. Clinical signs of cytauxzoonsis improved by 24 d postinfestation in all but one cat, with survivors remaining parasitemic and subclinically infected with C. felis. Unengorged A. americanum and D. variabilis were collected from wild habitats to determine the minimum infection rate of C. felis in ticks from an enzootic area. Infection of C. felis was found only in wild-collected A. americanum. The minimum infection rate of C. felis in A. americanum was 0.5% (one of 178) in males, 0.8% (three of 393) in nymphs, and 1.5% (three of 197) in females. We found no wild-collected D. variabilis infected with C. felis. Our results confirm that A. americanum is a primary vector of C. felis.

Inoculation of white-tailed deer (Odocoileus virginianus) with Ap-V1 Or NY-18 strains of Anaplasma phagocytophilum and microscopic demonstration of Ap-V1 In Ixodes scapularis adults that acquired infection from deer as nymphs.

Four white-tailed deer were inoculated with either the Ap-V1 or NY-18 strain of Anaplasma phagocytophilum. Ixodes scapularis nymphs were then allowed to acquistion feed on the inoculated deer and molt to adults. Only an Ap-V1 infected deer was infected persistently and able to infect nymphal Ixodes scapularis. Molted adult ticks maintained Ap-V1 infection as demonstrated by PCR and microscopy. We report, for the first time, a morphologic description of A. phagocytophilum in I. scapularis.