Although these animals will eventually develop diabetes which is corrected with IL-4 injection, the hypoglycemia in the IUGR animals does not appear until adulthood [11], and thus, the effects on myelination in this model were direct and not due to rescue of the metabolic syndrome. injury. IUGR induces an exaggerated Th2 response in the developing rat brain, including upregulation of several Th2 cytokines. Of these, IL-4 is usually significantly increased during the period corresponding to strong developmental myelination. We show that neutralizing IL-4 antibody therapy given in the newborn period ameliorates inflammation and restores myelin protein expression and oligodendrocyte cell number in the IUGR brain to control levels, demonstrating a novel role for Th2 responses and IL-4 in IUGR and white matter injury. In addition, IL-4 directly affects oligodendrocytes in vitro decreasing differentiation. Conclusions In this study, we have identified inflammation as a factor in the decrease in myelin seen in an animal model of IUGR. IL-4, an inflammatory protein often thought to be protective in the adult, is specifically increased, and treatment of these animals to prevent this increase ameliorates white matter damage. Our results suggest that the immune system plays a role in IUGR that is different in the perinatal period than in the adult and preventing this exaggerated Th2 response may be a potential therapeutic target. test. Western blotting Cell extracts were prepared from PD14 whole rat brain (excluding the hind brain) in ice-cold tissue extraction buffer as previously described [5], followed by centrifugation at 14,000?rpm at 4?C for 30?min. Protein concentrations of collected supernatants were determined by a NanoDrop spectrophotometer. Ten to 25?g of protein was loaded into each lane of 4C12% Bis-Tris gradient gel for separation. For detection of PLP, gels DMXAA (ASA404, Vadimezan) were run under non-reducing conditions due to antibody specificity. A broad spectrum molecular weight ladder was run on each gel. Following separation, proteins were transferred onto Millipore Immobilon-FL membranes and blocked in TBS with 0.1% Tween-20 (PBST) and 5% milk for 30 min at 4?C. Membranes were incubated overnight at 4?C with primary antibodies in TBST +?5% BSA. Membranes were incubated with the following primary antibodies: anti-myelin basic protein (MBP, rat hybridoma supernatant, 1:1000), anti-proteolipid protein (PLP, rat hybridoma supernatant, 1:1000), anti-CNP (Abcam, 1:1000), and anti-GFAP (rat hybridoma, 1:5000). All secondary antibodies were conjugated with IrDye at DMXAA (ASA404, Vadimezan) either 680 or 800 (LI-COR, Odyssey) and used at 1:10,000. Membranes were washed with PBST, and incubated with corresponding antigen-specific fluorescent probe-conjugated secondary antibodies (1,10,000 DMXAA (ASA404, Vadimezan) dilution) in TBST +?5% BSA. The membranes CD117 were imaged using Odyssey (Li-Cor). Blots were additionally probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:8000, Chemicon International) or tubulin (1:10,000, Sigma, St. Louis, MO) as a loading control for protein quantification. Bands of interest were specified to determine pixel intensities for each treatment using Licor Odyssey Software (Lincoln Nebraska), and the band intensities were normalized to loading controls to ensure equal loading. Statistical significance for the protein quantification was calculated using Students test. Neutralizing IL-4 therapy Control and IUGR animals were injected subcutaneously with 0.05?g of purified mouse anti-rat IL-4 antibody (BD Pharmingen) or PBS (Fisher BioReagents) daily from postnatal days 1C5 as previously described [11]. At postnatal day 14, either rats were perfused for immunohistochemistry or brains were collected and frozen for immunoblotting. Cell culture generation and treatment To generate DMXAA (ASA404, Vadimezan) cultures of purified OPCs from newborn rats, a mixed population of cells was harvested from the neonatal brain and seeded on 75-mL polylysine-coated flasks containing Neurobasal medium (Invitrogen, Life Technologies, Grand Island,.