Neuroprotective Effects of Delayed TGF-ß1 Receptor Antagonist Administration On Perinatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic (HI) brain injury in neonatal encephalopathy triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the primary damage. The cellular processes mediating this prolonged neurodegeneration in HI injury are not sufficiently understood. Consequently, current therapies are not fully protective. In a recent study, we found significant improvements in neurologic outcomes when a small molecule antagonist for Activin-Like Kinase 5 (ALK5), a transforming growth factor beta (TGF-ß) receptor was used as a therapeutic in a rat model of moderate term HI. Here, we have extended those studies to a mouse preterm pup model of HI. For these studies, postnatal day 7 (P7) CD1 mice of both sexes were exposed to 35-40 minutes of HI. Beginning 3 days later, SB505124, the ALK5 receptor antagonist, was administered systemically through intraperitoneal injections performed every 12 hours for 5 days. When evaluated 23 days later, SB505124-treated mice had ~2.5-fold more hippocampal area and ~2-fold more thalamic tissue. Approximately ~90% of the ipsilateral hemisphere (ILH) was preserved in the SB505124-treated HI mice compared to the vehicle-treated HI mice, where the ILH was ~60% of its normal size. SB505124 also preserved the subcortical white matter. SB505124 treatment preserved levels of aquaporin-4 and n-cadherin, key proteins associated with blood-brain-barrier function. Importantly, SB505124 administration improved sensorimotor function as assessed by a battery of behavioral tests. Altogether these data lend additional support to the conclusion that SB505124 is a candidate neuroprotective molecule that could be an effective treatment for HI-related encephalopathy in moderately injured preterm infants.

Moderately Inducing Autophagy Reduces Tertiary Brain Injury after Perinatal Hypoxia-Ischemia.

Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)ß1 is highly elevated following HI and that delivering an antagonist for TGFß receptor activin-like kinase 5 (ALK5)-SB505124-three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.

Neuroregenerative and protective functions of Leukemia Inhibitory Factor in perinatal hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic encephalopathy remains the most important neurological problem of the newborn. Delays in diagnosing perinatal brain injuries are common, preventing access to acute therapies. Therefore, there is a critical need for therapeutic strategies that are beneficial when delivered beyond 24 h after birth. Here we show that Leukemia Inhibitory Factor (LIF) functions as an essential injury-induced neurotrophic cytokine in the CNS and that non-invasively administering LIF as late as 3 days after a hypoxic-ischemic insult improves neurological function. Using a mouse model of late preterm brain injury we show that astroglial and microglial/macrophage reactivity to hypoxia-ischemia was diminished at 3 days of recovery, but then exacerbated at 2 weeks of recovery in LIF haplodeficient mice. There also were significantly more CD68+/Iba-1+ cells in the ipsilateral striatum in LIF-Het mice compared to WT mice at 2 weeks of recovery. This desynchronized glial response was accompanied by increased neuronal cell death in the striatum and neocortex (Fluorojade C), hypomyelination (reduced MBP staining and thinner external capsule), increased extent of brain damage (Nissl) and diminished neurological function on sensorimotor tests. To our surprise, injured LIF-Het mice had ~7-fold higher IGF-1 levels than injured WT mice at 3 days after HI injury. Intranasally administered LIF activated the Jak-Stat-3 pathway both within the subventricular zone and the neocortex at 30 min after administration. When delivered with a delay of 3 days after the insult, intranasal LIF reduced the extent of brain injury by ~60%, attenuated astrogliosis and microgliosis in striatum, improved subcortical white matter thickness, increased numbers of Olig2+ cells in corpus callosum and improved performance on sensorimotor tests at 2 weeks of recovery. These studies provide key pre-clinical data recommending LIF administration as a neuroprotectant and regenerative cytokine and they highlight the feasibility of pursuing new therapeutics targeting the tertiary phase of neurodegeneration for hypoxic-ischemic encephalopathies.