UCSF

The result of a deprivation of oxygen and glucose to the brain, hypoxic-ischemic encephalopathy (HIE), remains the most common cause of death and disability in human neonates globally and is mediated by glutamate toxicity and inflammation. We have previously shown that the enzyme glutamate carboxypeptidase (GCPII) is overexpressed in activated microglia in the presence of inflammation in fetal/newborn rabbit brain. We assessed the therapeutic utility of a GCPII enzyme inhibitor called 2-(3-Mercaptopropyl) pentanedioic acid (2MPPA) attached to a dendrimer (D-2MPPA), in order to target activated microglia in an experimental neonatal hypoxia-ischemia (HI) model using superoxide dismutase transgenic (SOD) mice that are often more injured after hypoxia-ischemia than wildtype animals. SOD overexpressing and wild type (WT) mice underwent permanent ligation of the left common carotid artery followed by 50 min of asphyxiation (10% O₂) to induce HI injury on postnatal day 9 (P9). Cy5-labeled dendrimers were administered to the mice at 6 h, 24 h or 72 h after HI and brains were evaluated by immunofluorescence analysis 24 h after the injection to visualize microglial localization and uptake over time. Expression of GCPII enzyme was analyzed in microglia 24 h after the HI injury. The expression of pro- and anti-inflammatory cytokines were analyzed 24 h and 72 h post-HI. Brain damage was analyzed histologically 7 days post-HI in the three randomly assigned groups: control (C); hypoxic-ischemic (HI); and HI mice who received a single dose of D-2MPPA 6 h post-HI (HI+D-2MPPA). First, we found that GCPII was overexpressed in activated microglia 24 h after HI in the SOD overexpressing mice. Also, there was an increase in microglial activation 24 h after HI in the ipsilateral hippocampus which was most visible in the SOD+HI group. Dendrimers were mostly taken up by microglia by 24 h post-HI; uptake was more prominent in the SOD+HI mice than in the WT+HI. The inflammatory profile showed significant increase in expression of KC/GRO following injury in SOD mice compared to WT at 24 and 72 h. A greater and significant decrease in KC/GRO was seen in the SOD mice following treatment with D-2MPPA. Seven days after HI, D-2MPPA treatment decreased brain injury in the SOD+HI group, but not in WT+HI. This reduced damage was mainly seen in hippocampus and cortex. Our data indicate that the best time point to administer D-2MPPA is 6 h post-HI in order to suppress the expression of GCPII by 24 h after the damage since dendrimer localization in microglia is seen as early as 6 h with the peak of GCPII upregulation in activated microglia seen at 24 h post-HI. Ultimately, treatment with D-2MPPA at 6 h post-HI leads to a decrease in inflammatory profiles by 24 h and reduction in brain injury in the SOD overexpressing mice.