Similar levels of most cytokines were seen after both Pam3CSK4 and LPS treatment, except for IL-1? and IL-6, which was only significantly elevated following TLR2 agonist stimulation but not LPS stimulation. Of note, such observations may be consistent with the polarization of neonatal mononuclear cells towards Sulfidefluor 7 AM relatively high TLR2-mediated IL-6 production. Whether such differences in cytokine responses between Pam3CSK4 and LPS treatment contributed to the differences in microglia activation between these two treatments will be the subject of future investigation. Interestingly, IL-1? is known to sensitize excitotoxic neonatal brain injury and blocking of the IL-1? receptor protects the immature brain from hypoxic-ischemic brain damage. We did not observe differences in markers of proliferation or apoptosis at least not at PND 12 and 53, but decreased mature neuronal number suggests that effects of Pam3CSK4 on cell survival may have occurred at a time point prior to that examined. The liver and spleen play a central role in immune responses and the liver is crucial in metabolizing microbial constituents such as Pam3CSK4. Thus the transient enlargement of the spleen and liver in Pam3CSK4 treated mice may indicate an acute reaction of the adaptive immune system and attempts to remove Pam3CSK4 in the blood. Although our study demonstrates that repeated, high-dose, systemic administration of a TLR2 agonist can lead to CNS injury, it is important to note that these effects are likely contextdependent. Indeed, vaccines containing TLR2 agonists, including intradermal bacille Calmette-Guerin and certain formulations of the intramuscular Haemophilus influenzae type b vaccine, have been safely and effectively administered to millions of infants. This underscores the importance of context, including route, frequency, and dose of administration when considering the impact of TLR agonists in injury models. In conclusion, we found that systemic administration of a TLR2 agonist to neonatal mice caused transient gray and white matter injury in both the cerebrum and cerebellum. This suggests that engagement of the TLR2 pathway can have detrimental effects on the developing brain, and may play a role in neonatal brain injury Fenobam associated with bacterial sepsis. However, neonatal brain injury is often multifactorial, and TLR2 agonist effects may interact with other exposures such as hypoxia/ischemia and/or be involved in a broader inflammatory response following Gram-positive bacterial exposure.