Relative degrees of injury within the dose range were 0

Relative degrees of injury within the dose range were 0.39 0.07 (= 9), 0.56 0.05 (= 5), 0.74 0.02 (= 7), 0.94 0.03 (= 5) and 1.38 0.12 (= 5) versus control [1.00 0.08 (= 9)]. because of its anti-inflammatory and neuroprotective results when provided after disease starting point, abrogated IgG-mediated neuroprotection and related microglial effects when given before injury. In contrast, E-prostanoid receptor subtype 2 (EP2) activation, which served as an exemplary paracrine stimulus like the one expected from neuronal activity, amplified IgG-mediated increased microglial recycling endocytosis and TNF- production. Furthermore, like monomeric IgG these EP2 related effects took days to be effective, suggesting both were adaptive anabolic effects consistent with those seen from other long-term preconditioning stimuli requiring protein synthesis. The data provide the first evidence that brain monomeric IgG at physiological levels can have signaling function via enhanced recycling endocytosis/TNF- production from microglia unassociated with disease and that these IgG-mediated changes may be a means by which paracrine signaling from neuronal activity influences microglia to evoke neuroprotection. The data provide further support that low-level proinflammatory neural immune signaling unassociated with disease enhances Proc brain function. = 5; 300C320 gm) were anesthetized with intraperitoneal pentobarbital (50 mg/kg) and placed in a standard stereotaxic unit. CSF (60C100 l/animal) was withdrawn at the atlanto-occipital junction via a 26 gauge needle and tuberculin syringe mounted on a Narishige micromanipulator from a sterile exposure of the dorsal neck at the occiput-upper cervical spine. Samples were stored at ?80C until quantification for IgG via microsphere-immunoassay. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Chicago. BETP Culture preparation and maintenance. Slice cultures are widely accepted experimental models and were prepared as previously described (Kunkler and Kraig, 1997; Kunkler et al., 2004, 2005). However, after 4 d before use. Primary microglia cultures were prepared as previously described (Caggiano and Kraig, 1999) using Dulbecco’s Modified Medium (#D5976; Sigma) plus 10% fetal bovine serum (#10082C147; Invitrogen) and 10 g/ml gentamicin (Invitrogen) (DMEM-10). Experimental manipulations. We examined whether BETP IgG could be neuroprotective by exposing slice cultures to nonspecific pooled rat IgG (#012C000-003) or nonspecific pooled F(ab)2 fragment (#115C006-003) in serum-free media (Jackson ImmunoResearch) for 3 d. IgG doses were 0.2, 2.0, 20, 200 or 2000 g/ml given for 3 d before and during injury (i.e., chronic exposure). The impact of acute exposure (i.e., 6 h before injury and immediately after injury) to IgG was also examined as well as prolonged exposure for 17 d. Exposure to F(ab)2 (3 d) was performed at IgG doses (0.2, 2.0, 20 g/ml) protective against excitotoxic injury. Serum-free media containing treatments were refreshed on the third day before injury inductions. To discover whether immune complexes could influence potential effects from IgG, we exposed slice cultures for 3 d to immune complexes formed from equal volumes of chicken-ovalbumin (2 g/ml, #A2512; Sigma) and affinity purified rabbit anti-ovalbumin IgG (40 g/ml, #ROL-30A; Immunology Consultants Laboratory) mixed and incubated at 36C for 30 min before use. Sodium azide was removed from rabbit anti-ovalbumin IgG via dialysis. We tested whether IgG effects were related to TNF- by use of soluble TNF receptor 1 (sTNFR1, 200 ng/ml, BETP #425-R1C050; R & D Systems) to abrogate TNF- signaling. Recombinant rat TNF- (#510-RT; R & D Systems) was applied to slice cultures (100 pg/ml) to mimic TNF- secretion from IgG exposure. Minocycline (10 m, #M9511; Sigma) was used to inhibit microglial activation (Lai and Todd, 2006). Sham experiments consisted of heat-inactivated IgG, F(ab)2 fragments or recombinant (i.e., sTNFR1 and TNF-) proteins [made by exposure to 95C (10 min) followed by ice (10 min) before use]. This was done to denature experimental proteins, and so remove their potential effects. However, production of recombinant proteins (i.e., TNF- and sTNFR1 in experiments here) involved use of 0.001; = 1.00) reductions in injury at 2.0 and 20 (shown) g/ml [with relative injury levels of 0.89 0.06 (= 13), 0.67 0.04 (= 13), 0.55 0.05 (= 13), 1.04 0.06 (= 21) and 0.92 0.04 (= 20) vs control (1.00 + 0.03; = 28)]. Heat-inactivated (HI) IgG (2.0 and 20 g/ml) had BETP no protective effect against NMDA [with equal to 0.58 (differences in means) and relative injury levels of 0.92 0.07 (n=11) and 0.92 0.07 (= 27) vs control (1.00 0.03; = 21)]. Similarly, monomeric IgG-triggered neuroprotection against OGD-induced excitotoxic injury showed a similar U-shaped response in slice cultures. 0.001; = 1.00) reductions in injury at 0.2, 2.0 (shown) and 20 (= 8), 0.49 0.05 (= 11), 0.0.59 0.08 (= 10), 0.95 0.05 (= 6), and 1.02 0.04 (= 6) vs control (1.00 0.02; = 11)]. HI IgG (0.2 and 2.0 g/ml) had no protective effect against OGD [with = 1.00 (differences in means) with relative injury levels of 1.03 0.06 (= 15), 1.00 0.04 (= 15) vs control (1.00 0.06;.