Current management for CP primarily focuses on rehabilitation and improving quality of life. Therapeutic approaches being explored include hypothermia for perinatal asphyxia, and stem cell infusion for CP (38
ID NCT01147653, NCT01072370). A key challenge in evaluating therapies for CP has been the paucity in animal models demonstrating the phenotype as seen in humans. The parallels in the timing of white matter development along with microglial presence in the human and rabbit brain, makes rabbit models of fetal brain injury more representative of CP in humans (27
). The presence of activated microglia in the periventricular white matter, oxidative injury, impaired myelination and neuronal loss seen in this model are consistent with histological findings seen in post-mortem brain of patients with PVL (8
). In addition, we find a predominance of hindlimb involvement in these animals which is similar to the increased incidence of diparetic CP (involving lower extremities) in children born preterm to mothers with placental inflammation or infection (6
We showed that systemic administration of hydroxyl-terminated PAMAM dendrimer resulted in their selective accumulation in activated microglia and astrocytes only in newborn kits with CP. We attribute this increased brain and cell uptake in CP kits, to impairment of the BBB in the periventricular region, presumably leading to the increased permeability to dendrimers. This is consistent with previous reports of PAMAM dendrimers crossing the blood-brain-tumor barrier in models of malignant gliomas (39
). An increase in the number of activated microglia and astrocytes, with enhanced phagocytic abilities under pathological conditions, may further facilitate the selective cellular localization of dendrimers in kits with CP (40
). Technical and ethical considerations make direct evaluation of the BBB difficult in patients. However, studies in newborn animal models of white matter injury have shown increased permeability of the BBB in the presence of inflammation (41
). Impairment of the BBB, in the presence of neuroinflammation, has also been reported in stroke, multiple sclerosis and in Alzheimer’s disease (42
). Passive targeting with dendrimers may facilitate delivery of therapeutics to neuroinflammation in these indications.
Intravenous administration of a single 10 mg/kg dose of D-NAC resulted in a significant improvement in neuronal injury and motor function in CP kits (movie S3
), while free NAC at 100 mg/kg did not, suggesting the importance of targeted drug delivery in the treatment of ongoing neuroinflammation. Even though free NAC_100 showed some efficacy in attenuating inflammation and oxidative injury in the brain, the improvement did not translate to myelination, neuronal counts or motor function. Moreover, the improvements seen with NAC_100 were similar to that seen with D-NAC at 1% of the dose (D-NAC_1). We speculate that this could be due to several factors, including poor bioavailability of free NAC (43
), improved uptake and efficacy of D-NAC when compared to free NAC in activated microglia, as shown previously in
), delivery of a higher drug-payload to the target cells (activated microglia and astrocytes) by the dendrimer in vivo, and decreased toxicity of the drug to neurons when conjugated with the dendrimer.
In the presence of inflammation and oxidative stress, depletion of GSH is one of the mechanisms by which the neuroprotective function of astrocytes is compromised (12
). Regulated neuro-glial transport of glutathione and cysteine from astrocytes to neurons may play a role in neuroprotection (44
). Hence replenishing GSH specifically in astrocytes by D-NAC may help improve neuronal survival. In addition, excess extracellular L-cysteine concentrations have been shown to result in neuronal degeneration by NMDA (N
-methyl-D-aspartate) mediated-excitoxicity both in vitro
and in vivo
). Therefore, targeted delivery of the drug to activated microglia and astrocytes can not only help attenuate inflammation but may also prevent excess extracellular levels of L-cysteine produced from NAC that may be toxic to neurons and oligodendrocytes in the immature brain. A therapeutic response was not seen upon treatment with dendrimer alone, which indicates that the dendrimer acts as a drug delivery vehicle. Although PAMAM dendrimers are not yet approved for clinical use, there are several pre-clinical studies involving them (16
. We use hydroxyl-terminated PAMAM dendrimers with a good safety profile in newborn kits, which may enable translation. In humans, since the exact time of the perinatal brain injury may vary, multiple injections of D-NAC, or sustained release formulations may be needed for effective therapy. Future longitudinal studies focusing on long term effectiveness of this therapy up to adulthood will facilitate clinical translation. The platform described herein to target activated microglia and astrocytes has broad implications for the treatment of neurological diseases given the growing body of evidence that neuroinflammation plays a key role in the pathogenesis of disorders such as multiple sclerosis, Alzheimer’s disease, and stroke. A similar therapeutic response with dendrimer-based targeting is also seen in models of retinal degeneration (19
This work demonstrates that targeted attenuation of ongoing neuroinflammation can have significant implications for the treatment of maternal intrauterine infection and inflammation induced brain injury, which leads to disorders such as cerebral palsy. The effectiveness of the dendrimer-NAC treatment, administered in the postnatal period for a prenatal insult, suggests a new window of opportunity for treatment of CP after birth in humans. Early detection of neuroinflammation using non-invasive, in vivo imaging techniques, such as PET and MRI, can help in identifying patients at high risk for developing motor deficits in the newborn period (26
). Targeted therapy for attenuation of neuroinflammation in at-risk patients, delivered at an early stage after birth, can potentially arrest or prevent the development of motor and cognitive deficits associated with perinatal brain injury and cerebral palsy. Using dendrimers to deliver drugs to activated microglia and astrocytes may eventually provide a versatile platform for the treatment of other neuroinflammatory disorders.