Age-Dependent Effects of ALK5 Inhibition and Mechanism of Neuroprotection in Neonatal Hypoxic-Ischemic Brain Injury.

Neonatal encephalopathy due to hypoxic-ischemic (HI) brain injury triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the initial insult. In a recent set of studies, we evaluated the therapeutic efficacy of a small molecule antagonist for ALK5 (activin-like kinase 5 ), TGF-ß receptor in a rat model of moderate perinatal HI and found significant improvements in neurologic outcomes. Here, we have extended those studies to evaluate the efficacy of delayed TGF-ß receptor antagonism on postnatal day (P) 6 and P9 HI rat pups with and without hypothermia. The ALK5 receptor antagonist SB505124 was administered systemically by osmotic pump beginning 3 days following HI. Extending our earlier data set that showed protection of the hippocampus in P6 pups treated with SB505124, these animals sustained less damage to their hippocampi and had improved performance on the Morris water maze (MWM) when tested on P60 versus vehicle-treated HI animals. By contrast, SB505124 did not improve sensorimotor deficits and exacerbated hippocampal and thalamic volume loss when administered 3 days after HI to P9 pups. SB505124-treated rats injured on P9 tended to perform worse than their vehicle-treated counterparts on MWM, and SB505124 treatment did not preserve hippocampal or thalamic neurons in P9 pups when combined with hypothermia. To elucidate the mechanism whereby ALK5 inhibition reduced neuronal death in the P6 HI model, we assessed levels of autophagy markers in neurons of the neocortex, hippocampus, and thalamus, and in the subcortical white matter, and found that SB505124 increased numbers of autophagosomes and levels of lipidated LC3 (light chain 3), a key protein known to mediate autophagy. Altogether, our results demonstrate that there is a dynamic switch in the CNS response to TGF-ß1 that occurs around P9 in rats where TGF-ß signaling inhibition worsens functional outcomes. This response is similar to the outcome of antagonizing TGF-ß signaling in adult stroke and other CNS disease models. We conclude that attenuating TGF-ß1 signaling will likely be an effective treatment for HI-related encephalopathy in moderately preterm infants, offering protection of the neocortex, hippocampus, and thalamus with enhanced cerebral autophagy contributing to the decrease in the extent of progressive neuronal cell death.

Mechanisms of mouse neural precursor expansion after neonatal hypoxia-ischemia.

Neonatal hypoxia-ischemia (H-I) is the leading cause of brain damage resulting from birth complications. Studies in neonatal rats have shown that H-I acutely expands the numbers of neural precursors (NPs) within the subventricular zone (SVZ). The aim of these studies was to establish which NPs expand after H-I and to determine how leukemia inhibitory factor (LIF) insufficiency affects their response. During recovery from H-I, the number of Ki67(+) cells in the medial SVZ of the injured hemisphere increased. Similarly, the number and size of primary neurospheres produced from the injured SVZ increased approximately twofold versus controls, and, upon differentiation, more than twice as many neurospheres from the damaged brain were tripotential, suggesting an increase in neural stem cells (NSCs). However, multimarker flow cytometry for CD133/LeX/NG2/CD140a combined with EdU incorporation revealed that NSC frequency diminished after H-I, whereas that of two multipotential progenitors and three unique glial-restricted precursors expanded, attributable to changes in their proliferation. By quantitative PCR, interleukin-6, LIF, and CNTF mRNA increased but with significantly different time courses, with LIF expression correlating best with NP expansion. Therefore, we evaluated the NP response to H-I in LIF-haplodeficient mice. Flow cytometry revealed that one subset of multipotential and bipotential intermediate progenitors did not increase after H-I, whereas another subset was amplified. Altogether, our studies demonstrate that neonatal H-I alters the composition of the SVZ and that LIF is a key regulator for a subset of intermediate progenitors that expand during acute recovery from neonatal H-I.

Intranasal administration of aTf protects and repairs the neonatal white matter after a cerebral hypoxic-ischemic event.

Our previous studies showed that the intracerebral injection of apotransferrin (aTf) attenuates white matter damage and accelerates the remyelination process in a neonatal rat model of cerebral hypoxia-ischemia (HI) injury. However, the intracerebral injection of aTf might not be practical for clinical treatments. Therefore, the development of less invasive techniques capable of delivering aTf to the central nervous system would clearly aid in its effective clinical use. In this work, we have determined whether intranasal (iN) administration of human aTf provides neuroprotection to the neonatal mouse brain following a cerebral hypoxic-ischemic event. Apotransferrin was infused into the naris of neonatal mice and the HI insult was induced by right common carotid artery ligation followed by exposure to low oxygen concentration. Our results showed that aTf was successfully delivered into the neonatal HI brain and detected in the olfactory bulb, forebrain and posterior brain 30 min after inhalation. This treatment successfully reduced white matter damage, neuronal loss and astrogliosis in different brain regions and enhanced the proliferation and survival of oligodendroglial progenitor cells (OPCs) in the subventricular zone and corpus callosum (CC). Additionally, using an in vitro hypoxic model, we demonstrated that aTf prevents oligodendrocyte progenitor cell death by promoting their differentiation. In summary, these data suggest that iN administration of aTf has the potential to be used for clinical treatment to protect myelin and to induce remyelination in demyelinating hypoxic-ischemic events in the neonatal brain.

Delayed ALK5 inhibition improves functional recovery in neonatal brain injury.

Neuroinflammation subsequent to developmental brain injury contributes to a wave of secondary neurodegeneration and to reactive astrogliosis that can inhibit oligodendrocyte progenitor differentiation and subsequent myelination. Here we evaluated the therapeutic efficacy of a small molecule antagonist for a TGFß receptor in a model of moderate perinatal hypoxia-ischemia (H-I). Osmotic pumps containing SB505124, an antagonist of the type 1 TGFß1 receptor ALK5, or vehicle, were implanted three days after H-I induced at postnatal day 6. Perinatal H-I induced selective neuronal death, ventriculomegaly, elevated CNS levels of IL-6 and IL-1α, astrogliosis, and fewer proliferating oligodendrocyte progenitors. Myelination was reduced by ∼50%. Anterograde tracing revealed extensive axonal loss in the corticospinal tract. These alterations correlated with functional impairments across a battery of behavioral tests. All of these parameters were brought back towards normal levels with SB505124 treatment. Notably, SB505124 preserved neurons in the hippocampus and thalamus. Our results indicate that inhibiting ALK5 signaling, even as late as three days after injury, creates an environment that is more permissive for oligodendrocyte maturation and myelination producing significant improvements in neurological outcome. This new therapeutic would be especially appropriate for moderately preterm asphyxiated infants, for whom there is presently no FDA approved neuroprotective therapeutic.

Apotransferrin-induced recovery after hypoxic/ischaemic injury on myelination.

We have previously demonstrated that aTf (apotransferrin) accelerates maturation of OLs (oligodendrocytes) in vitro as well as in vivo. The purpose of this study is to determine whether aTf plays a functional role in a model of H/I (hypoxia/ischaemia) in the neonatal brain. Twenty-four hours after H/I insult, neonatal rats were intracranially injected with aTf and the effects of this treatment were evaluated in the CC (corpus callosum) as well as the SVZ (subventricular zone) at different time points. Similar to previous studies, the H/I event produced severe demyelination in the CC. Demyelination was accompanied by microglial activation, astrogliosis and iron deposition. Ferritin levels increased together with lipid peroxidation and apoptotic cell death. Histological examination after the H/I event in brain tissue of aTf-treated animals (H/I aTF) revealed a great number of mature OLs repopulating the CC compared with saline-treated animals (H/I S). ApoTf treatment induced a gradual increase in MBP (myelin basic protein) and myelin lipid staining in the CC reaching normal levels after 15 days. Furthermore, significant increase in the number of OPCs (oligodendroglial progenitor cells) was found in the SVZ of aTf-treated brains compared with H/I S. Specifically, there was a rise in cells positive for OPC markers, i.e. PDGFRα and SHH(+) cells, with a decrease in cleaved-caspase-3(+) cells compared with H/I S. Additionally, neurospheres from aTf-treated rats were bigger in size and produced more O4/MBP(+) cells. Our findings indicate a role for aTf as a potential inducer of OLs in neonatal rat brain in acute demyelination caused by H/I and a contribution to the differentiation/maturation of OLs and survival/migration of SVZ progenitors after demyelination in vivo.