Neuroprotective Effects of Delayed TGF-β1 Receptor Antagonist Administration on Perinatal Hypoxic-Ischemic Brain Injury

Abstract 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-β) 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 CD1 mice of both sexes were exposed to 35–40 min of HI. Beginning 3 days later, SB505124, the ALK5 receptor antagonist, was administered systemically through intraperitoneal injections performed every 12 h 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.


Introduction
Neonatal hypoxia-ischemia (HI) and pediatric stroke together occur in over 6 in 1,000 infants annually.Most newborns survive this adverse event, yet 50-80% of them have neurodevelopmental sequelae such as epilepsy and cognitive impairments for their lifetime [1].HI in preterm infants also is a serious health condition that has been underappreciated due to difficulties in diagnosing this condition [2].Treatments for cerebral HI are insufficient at preventing brain damage.Currently, term infants are given therapeutic hypothermia whereas preterm infants are only given magnesium sulfate [3].In both cases, these interventions need to be administered within the first few hours of the initial injury and neither provide complete neuroprotection [4,5].Therapeutic hypothermia is approved for the treatment of moderate neonatal encephalopathy and has been shown to improve survival if administered within the first 6 h of life [6,7].However, the therapy is not perfect; it presents complications and offers limited functional recovery.The therapy is also contraindicated in preterm infants, thus preterm infants often face lifelong learning problems and other challenges.In a recent review, we defined 3-phases of neurodegeneration after HI: acute, secondary, and tertiary [8].The acute period can be defined as those events that occur during the first 6 h after cerebral HI where events lead to excitotoxic cell deaths [9].During the secondary period of injury, which we defined as those events that occur from 7 to 48 h after the injury, oxidative stress damages proteins and lipids necessary for cell survival, and signaling cascades triggered by cytokines, lead to apoptotic cell death [10][11][12][13][14][15].During this period of secondary injury, there is a second wave of cerebral energy failure that has been observed in both humans and in animal models that contributes to additional secondary cell deaths [16][17][18][19].Over the next few days to weeks, the injury appears to stabilize but depending upon the extent of injury, as well as a variety of other factors, more cells become damaged contributing to progressive neurodegeneration in this third phase that we defined as the tertiary phase.To date, there are no therapeutics that can reproducibly target the tertiary phase of brain injury [20].Therefore, new therapies are desired to be used alone or combined with hypothermia to improve the course of brain development.
Our laboratory has documented production of the cytokine transforming growth factor beta-1 (TGF-β1) during recovery from cerebral HI in rat pups.TGF-β1 production following CNS injury is both multicellular and multiregional: TGF-β1 expression is elevated in neurons and in glia, and it may enter the brain parenchyma from the periphery by way of leaky vasculature.After a stroke, TGF-β1 is principally produced by activated microglia and macrophages as neurons, and astrocytes produce much less TGF-β1 than the activated microglia [6,21].Human postmortem samples from injured preterm infants confirm elevated TGF-β expression in microglia [21].However, the consequences of TGF-β1 induction in the context of injury are only beginning to be understood.
Current evidence suggests that both the extent and timing of TGF-β signaling are critical in determining whether TGF-β1 will aggravate inflammation or help resolve it [22].TGF-β1 may exert beneficial effects at the region of injury, but prolonged increases in signaling in the brain lead to exacerbated injury.This concept was initially illustrated in studies of reactive astrogliosis, where TGF-β1 expression was rapidly induced following CNS lesions and remained elevated within the glial scar for up to 14 days post-injury [23].TGF-β1 is a key mediator of glial scar formation as it induces astroglial cell proliferation, GFAP expression, and deposition of a dense extracellular matrix [23].Reactive astrogliosis may be beneficial in the early stages of injury by facilitating the removal of toxic substances in the local microenvironment and limiting lesion growth.However, in models of neonatal and adult stroke, functional recovery becomes inhibited once a permanent scar forms as it can restrict angiogenesis into the core of injury and severely limits axonogenesis, synaptogenesis, and remyelination [24].
Studies in our laboratory and others have identified TGF-β1 as a key cytokine that promotes delayed neurodegeneration.TGF-β1 activates activin-like kinase 5 (ALK5) to stimulate astrocyte proliferation and proinflammatory cytokine production [21,[25][26][27].Our laboratory showed that systemically administering an ALK5 antagonist, SB505124, for 7 days, beginning 3 days after induction of HI in postnatal day 6 rat pups, reduced the extent of astrogliosis, ventriculomegaly, and CNS levels of IL-6 and IL-1α while preserving neurons in the corticospinal tract, thalamus, and hippocampus by de-repressing autophagy [7].SB505124 treatment also restored myelination and enhanced sensorimotor function.To assess whether SB505124 would exert cross species neuroprotection and to enable future studies using genetically engineered mice, here we validated the efficacy of SB505124 in a neonatal mouse model of preterm HI.These studies were designed to test the hypothesis that administering a TGF-β receptor antagonist that targets the ATP-binding pocket of the ALK5 receptor during the subacute stage of recovery from perinatal HI will reduce the extent of neurodegeneration and improve neurological function.

Rodents
All experiments were performed in accordance with research guidelines set forth by Institutional Animal Care and Use Committee of Rutgers New Jersey Medical School (Protocol #999900841) and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (publication No. 80-23) revised in 1996 and the ARRIVE Delayed ALK5 Antagonism Protects the Immature Brain from HI guidelines.Following delivery, litter sizes were adjusted to 12 pups per litter, and efforts were made to ensure the number of each sex, and pup weights were equal and consistent.Animals were group housed and kept on a 12-h light:dark cycle with ad libitum access to food and autoclaved water.Mouse pups remained with the dam until the day of HI injury.

Neonatal Hypoxic-Ischemic Brain Injury
Cerebral HI was induced in 7-day-old mouse pups (postnatal day 7 [P7], day of birth = P0; mean body mass ~6 g), as a model of preterm injury [28].Briefly, CD1 mice were anesthetized with isoflurane (3-4% induction, 2-3% maintenance) prior to right common carotid artery cauterization.A special effort was made to carefully isolate the carotid artery without damaging other structures contained within the carotid sheath (i.e., internal jugular vein and vagus nerve).The neck incision was sutured with 4-0 surgical silk.Following a 1-h recovery period, the mice were transferred to jars that were suspended in a 36°C water bath.They were then exposed to 35-40 min of hypoxia using humidified 10% oxygen/ nitrogen balance.After this HI episode, they remained in the jars for another 15 min in room air and were then returned to the dams.Thus at all stages of the procedure, efforts were undertaken to maintain them normothermic.Sham mice were anesthetized for isolation of the right common carotid artery, but no cauterization was performed, to induce hypoxia without ischemia.Mice were randomly assigned to experimental groups after HI injury.Sample sizes per experiment were chosen to achieve sufficient statistical power with minimal numbers of animals based on pilot studies.

Western Blot Analyses
At 6 h, 12 h, and 6 days after initiating SB505124 treatment, microdissected brain tissue from the affected neocortex, striatum, and corpus callosum from the injured (ipsilateral) hemisphere was collected either as a single wedge of infarcted tissue or as microdissected regions when the damage was severe.The tissue was homogenized and then sonicated in RIPA lysis buffer with a tablet of 1% cOmplete ™ , Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 2 (Sigma-Aldrich).40 μg of denatured protein was loaded into a 4-12% Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad, CA, USA) and run with 5 µL of Amersham ECL Rainbow Marker as a molecular weight standard (GE Life Sciences, Pittsburgh, PA, USA) at 200 V, 130 mA, 90 W settings for an hour.Proteins were transferred onto nitrocellulose at 30 V, 180 mA, 8 W setting for an hour and 5 min and blocked for an hour with 5% bovine serum albumin in 1 × TBS/0.1% Tween before incubated with primary and secondary antibodies as detailed in Table 1.Membranes probed for primary antibodies overnight at 4°C were washed with 1 × TBS/0.1% Tween, incubated in HRP-linked secondary antibody, washed, and bands visualized using Western Lightning Chemiluminescence Reagent (PerkinElmer, Wellesley, MA, USA).Imaging for the antibodies was performed using a BioRad Chem-iDoc Imaging System combined with Image Lab software (Hercules, CA, USA).
Sensorimotor Testing Three weeks following HI injury, mice were subjected to a comprehensive battery of sensorimotor function tests aggregated into a modified neurological severity score (mNSS) as described originally by Chen et al. [29] and as modified as we described previously [30].Tests were conducted by an investigator blinded to the experimental groups.Starting 10 days after HI injury, each mouse was handled for approximately 5 min for 5 days in the morning to prevent confounding variables such as stress and anxiety on the day of behavioral testing.After 5 days, the mice were subjected to 3 days of pre-training.For the pre-training, mice were acclimated to a 1-cm width beam walking test, to a 0.7-cm inclined beam walking test, and to the horizontal ladder test.An escape platform was placed at the end of the tests with mouse chow and some of their cage bedding for the mice to feel comfortable.On the day of the behavioral tests, a comprehensive battery of sensorimotor function tests was used to produce an mNSS.Fine motor skills were then analyzed using a horizontal ladder with rungs spaced unevenly to increase complexity of the run [31,32].The average number of foot slips over three non-consecutive runs was calculated for each animal.All beam tasks and horizontal ladder walk tests were video recorded and were analyzed in a blinded fashion.In addition, the sticky paper test was performed, where a 5-mm diameter sticky paper was placed onto the contralateral forepaw and the time to remove it was measured [7,33].The mice Kanal/Levison also were tested using the hang test where the mouse pups were allowed to grab a horizontal metal rod with their forelimbs and their hang-time was measured.We also evaluated hind limb clasping where the mouse pups were held by their tail for 10 s and their hind limb contractions evaluated.Mouse pups, separated depending on their sexes at P20, were taken cage-bycage in order to perform the behavioral tests.

Histology
Three weeks following HI injury, mice were deeply anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) mix.After intracardiac puncture, they were perfused with culture medium and then with 3% paraformaldehyde in phosphate-buffered saline, pH 7.4.Whole brains were extracted and dehydrated in 70% ethanol and then embedded in paraffin.A total of twelve slides for forebrain and eight slides for hippocampal analysis were collected and three coronal sections were analyzed per animal to evaluate the gross damage produced by the HI insult.Forebrain hemisphere sizes were assessed from sections taken 0.2 mm from bregma, while hippocampal size was assessed from section −2.4 mm from bregma.White matter damage was assessed by Luxol fast blue staining, where after de-waxing the paraffin sections, they were incubated in 0.1% Luxol fast blue at 60°C for 15 min.After rinses in ethanol and distilled water, they were differentiated using 0.05% lithium chloride and then cleared using 70% ethanol [34].Sections also were stained with cresyl violet.Images were collected using an Olympus BX51 microscope (Center Valley, PA, USA), captured by a Q-imaging Retiga-2000R CCD camera (Surrey, BC, Canada) and acquired on PictureFrame software (Optronics; Goleta, GA, USA).Using ImageJ, a ventricle size index was calculated as the ratio of the IL ventricle to the whole ipsilateral hemisphere area; a hippocampus size index was calculated as the ratio of the ipsilateral hippocampus area to the contralateral hippocampus area.

Data Analysis and Statistics
Raw data from image analyses and behavioral tests were imported into Prism (GraphPad Software, La Jolla, CA, USA) for statistical analyses using one-way ANOVA followed by Tukey's post hoc intergroup comparison.Non-parametric results were analyzed by Kruskal-Wallis followed by Tukey's post hoc intergroup comparison.Graphs were produced in Prism, and error bars denote standard error of means.To randomize our selections, Research Randomizer software was used.

Evaluating SMAD2/3 Phosphorylation after HI
To determine the efficiency of SB505124 on the inhibition of canonical TGF-β signaling pathways and to establish the efficiency window of the injection interval, HI was performed on 7-day-old mouse pups.Three days after HI, 10 mg/kg of the ALK5 pharmacological inhibitor, SB505124, or vehicle, was administered once i.p.Brain levels of pSMAD2/3 were evaluated in homogenates from brain tissues extracted from the affected IL hemispheres 6 and 12 h after i.p. injection (Fig. 1).At 6 h after the administration of the inhibitor, there was a trend toward decreased levels of pSMAD2/3 in SB505124-treated HI animals compared to the sham animals (Fig. 1b and c, p = 0.07).Twelve hours after administrating SB505124, pSMAD2/3 levels were significantly decreased compared to both sham-and vehicletreated HI mice (Fig. 1d and e, p < 0.05).We did not see increased activation of canonical TGF-β signaling pathway in the vehicle-treated HI animals, suggesting an acute activation pattern in response to the injury [22].

Delayed Administration of SB505124 Preserves Components of the Blood-Brain Barrier
To elucidate the mechanism which the ALK5 pharmacological inhibitor, SB505124, reduced the tertiary neurodegeneration, we decided to look into the bloodbrain barrier (BBB) proteins; n-cadherin, aquaporin-4, and claudin-5 with respect to beta-actin levels.Three days after HI, 10 mg/kg of the SB505124, or vehicle, was administered twice a day for 2 days i.p.On P10 brain levels of the BBB proteins were evaluated in homogenates from brain tissues extracted from the affected IL hemispheres 5 days after injury (Fig. 2b).The relative levels of n-cadherin and aquaporin-4 were significantly reduced in the vehicle-treated HI animals compared to SB-treated HI animals (respectively, Fig. 2c and e, p < 0.001 and p < 0.05), whereas the relative levels of claudin-5 were similar among the groups (Fig. 2d, p > 0.05).

Delayed SB505124 Administration Reduces the Extent of Tertiary Neurodegeneration
Three days after HI, we administered SB505124 once every 12 h for 5 days to assess whether it would inhibit delayed neurodegeneration.Histopathological analyses of the lateral ventricles, hippocampus, thalamus, and neocortex were evaluated from cresyl violet stained brain sections 24 days after HI injury (postnatal day 30).HI caused significant ventricular dilation.The ventricle size index increased by 10.5-fold in HI vehicle group (1.38 ± 0.55 compared to the sham group 0.13 ± 0.06, p < 0.05, Fig. 3a,b).SB505124 treatment alleviated ventricle dilation reaching values no different from the shams (p > 0.05).A comparison of the hippocampi showed tissue preservation with SB505124 treatment.The IL hippocampus in SB505124-treated HI mice retained 61% ± 9.74% of its size compared to the contralateral side, while the IL hippocampus in vehicle-treated HI mice was 26.83% ± 8.25% of the contralateral side (Fig. 3c, f).Differences in hippocampal size were statistically significant between sham-and vehicle-treated HI animals (p < 0.0001) and Delayed ALK5 Antagonism Protects the Immature Brain from HI vehicle-and SB-treated HI animals (p < 0.05).Thalamic preservation was evident with the treatment.The IL thalamus in SB505124-treated HI mice retained 84.65% ± 8.21% of its size compared to the contralateral side, while the IL thalamus in vehicle-treated HI mice was 56.48% ± 8.56% of the contralateral side (Fig. 3d, f).Differences in thalamic size were statistically significant between shamand vehicle-treated HI animals (p < 0.001) and vehicle-and SB-treated HI animals (p < 0.01).A comparison of neocortices also showed tissue preservation with SB505124 treatment.The IL neocortex in SB505124-treated HI mice retained 87.91% ± 5.85% of its size compared to the contralateral side, while the IL thalamus in vehicletreated HI mice was 60.17% ± 13.65% of the contralateral side (Fig. 3e, f).Differences in neocortical size were statistically significant between sham-and vehicle-treated HI animals (p < 0.05), while having no significant difference between vehicle-and SB-treated HI animals (p < 0.1).
To assess white matter integrity, the area of myelinated external capsule and corpus callosum was analyzed on LFB-stained sections.HI injury caused aberrant white matter organization and loss of white matter integrity.Representative coronal sections taken near the striatum are illustrated in Fig. 4a-c.The area of the white matter in HI vehicle-treated animals versus sham-injured animals was 0.70 ± 0.13 compared to the SB505124-treated animals, which was 1.07 ± 0.09 (Fig. 4d).Differences in subcortical white matter area were statistically significant between sham-and vehicle-treated HI animals (p < 0.01) and between vehicle-and SB-treated HI animals (p < 0.05).

Delayed SB505124 Administration Improves
Sensorimotor Deficits after Perinatal HI As we were concerned that the systemic administration might have an adverse effect on the health of the subject, mice were weighed every 3 days.At 30 days of age, HI mouse pups treated with SB505124 had a higher mean body mass as compared to vehicle-treated HI group (Fig. 5b, p < 0.05) and no difference when compared to sham-operated animals (Fig. 5b, p > 0.05).
To determine whether SB505124 reduced the amount of brain damage, measurements of the gross hemispheric area were determined as a hemispheric ratio (injured ipsilateral area to contralateral area).This analysis revealed greater gross brain damage in vehicle-treated HI mice (p < 0.001 vehicle vs. sham and p < 0.05 vehicle vs. SB505124), while there was no significant difference in ratio between SB505124-treated HI animals and shaminjured animals (Fig. 5c).The mean preservation in vehicle-treated HI animals was 60%, whereas shamand SB505124-treated HI animals had a mean preservation of 96% and 84%, respectively.
Impressed with the extent of the tertiary neuroprotection observed with the gross brain damage, structural preservation of lateral ventricles, hippocampus, thalamus, neocortex, and the white matter, we wanted to determine whether long-term neurological functions were improved in HI mice. 3 days after HI injury, we administered SB505124 once every 12 h for 5 days, and at postnatal day 24, we subjected the HI mice to a battery of behavioral tests (Fig. 5a).Sensorimotor metrics were tabulated into a modified neurological severity score (mNSS) system, in which a higher score denotes poorer performance and consequently greater impairment (Table 2).Vehicle-treated HI mice scored significantly higher (2.9 ± 0.38) compared to SB505124 and sham control (1 ± 0.24 and 0.75 ± 0.25, respectively) (Fig. 5d).Sensorimotor deficits were also evaluated with the sticky paper test.Here, vehicle-treated HI mice took longer to remove the adhesive paper from the contralateral forepaw than the shams (122.9 s ± 25.58 s vs. 37.2 s ± 16.25 s, respectively, Fig. 5f, p < 0.05).Deficits in performing this test were corrected by SB505124 treatment (Fig. 5f) (51.25 s ± 25.58, p < 0.05).
Sensorimotor integration was tested using beam walking test and with the horizontal ladder test.The vehicletreated HI mice performed the worst, with the greatest number of foot slips per run on the horizontal 1.0 cm wide balance beam (0.73 ± 0.16) and horizontal ladder tests (0.77 ± 0.21) (Fig. 5f, g, e).SB505124-treated HI mice had significantly fewer foot slips than vehicle-treated HI mice on both tests (0.13 ± 0.07 and 0.27 ± 0.07, respectively, p < 0.05) and did not perform any differently compared to sham controls (Fig. 5f, g).A total of 28 animals (17 females and 11 males) were analyzed and no differences based on sex were observed in these behavioral analyses although they were not powered to reveal sex differences (data not shown).

Discussion
Here, we tested the hypothesis that administering a TGF-β receptor antagonist that targets the ATP-binding pocket of the ALK5 receptor during the subacute stage of recovery from perinatal HI will reduce the extent of neurodegeneration and improve neurological function.We show that administering SB505124 every 12 h starting 3 days after HI using a P7 mouse model: (1) decreased SMAD2/3 phosphorylation; (2) maintained levels of 3 key proteins required for BBB integrity; (3) prevented ventriculomegaly; (4) preserved hippocampal and thalamic volumes; (5) preserved the integrity of the corpus callosum and external capsule; and (6) improved performance on a battery of sensorimotor 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.

SB505124 Prevents Tertiary Neurodegeneration
Several clinical studies have contributed to the view that cerebral HI can trigger an evolving process, resulting in a worsening brain damage over time [8].For example, using T2-weighted magnetic resonance imaging, Barkovich et al. [35] showed that the largest changes after HIE occurred 6-10 days after injury, suggesting that the lesion continued to evolve over time.A similar diffusion-weighted imaging study showed that during recovery from HIE, the superficial lesions of the neocortex continued to expand over 3-4 days, recruiting deeper regions of the brain [36].Additional data reported by Shroff et al. [37] provided evidence that the injury in 1 out of 5 infants continues to progress as changes in diffusion-weighted imaging peak between 3 and 5 days of recovery.Similarly, animal imaging studies have provided strong support for delayed neurodegeneration after perinatal HI.Lama et al. [38] analyzed rat pups over the 1st month of recovery after perinatal HI and showed that between 1 and 3 days of recovery that there were areas of hyperintensity in the descending white matter tracts as well as decreases in the apparent diffusion co-efficient.
Staining for neurofilaments, they confirmed that there was a progressive loss of axons between 1 day and 4 months of recovery at the pontine level of the corticospinal tract.In a subsequent study, they showed that there was a ~2.5-fold increase in Fluoro-JadeC+ neurons between 1 day and 4 weeks of recovery [39].
In our earlier study in P6 rat pups where we delivered SB505124 by an osmotic pump between 3 and 10 days of recovery from HI, we found that SB505124 prevented the 17-fold increase in ventriculomegaly that was seen in vehicle-treated rat pups.This increase in ventricular volume is likely due to encephalomalacia rather than to an increase in intraventricular pressure, although our finding of preserved BBB components suggests that the latter mechanism could also be contributing to the ventriculomegally, as discussed below.Similar to the findings reported here, SB505124 treatment preserved the thalamus and hippocampus.Motor function was significantly improved that could be attributed to preserving upper motor neurons, as demonstrated by preserved numbers of anterograde-labeled corticospinal axons.That hemispheric volume was preserved for up to 3 weeks past the initial insult indicated ALK5 inhibition can confer long-term protection [7].
Analogous to that previous study, here we show that SB505124 is of benefit in this preclinical model of preterm HI brain injury, which is significant as there are presently no clearly effective therapeutics for this cohort of HI infants.Other molecules, such as erythropoietin and melatonin have been evaluated as therapeutics for perinatal HI with or without hypothermia [40][41][42].Both can be delivered as late as 24 h after the injury for the treatment of moderate neonatal hypoxic-ischemic encephalopathy; however, few other small molecule or peptide neuroprotectants can be delivered as late as 3 days after injury to neonates to prevent delayed CNS injury.One exception is leukemia inhibitory factor, which we have shown can be delivered intranasally beginning at 3 days of recovery where it exerts potent neuroprotective and antineuroinflammatory actions [30].

TGF-β Stimulates CNS Neuroinflammation
One hypothesis for the delayed neurodegeneration that is occurring is that the production of pro-inflammatory cytokines, and the subsequent CNS neuroinflammation causes a persistent and slow neuronal injury.Human postmortem samples from injured preterm infants confirm elevated TGF-β1 expression in microglia [21], and increased proinflammatory cytokines and microglial activation have been demonstrated up to 14 days after neonatal HI in rodent models [43,44].At the synaptic terminal, neuronal TGF-βsignaling is involved in complement-mediated microglial engulfment of presynaptic boutons [45].Therefore, elevated levels of TGF-β post-injury may activate aberrant microglial synapse pruning to cause synapse loss, which in turn would lead to loss of target-derived trophic support followed by retrograde neurodegeneration.Additionally, we and others have shown that TGF-β1 stimulates immature astrocytes to produce and secrete the pro-inflammatory mediators IL-6 and IL-1β [7,21,[25][26][27].TGF-β1 also has been shown to stimulate IL-6 secretion from pericytes [46].Numerous studies have shown that increased levels of IL-6 and IL-1 contribute to neurodegeneration after brain injuries [47,48].TGF-β Receptor Antagonism Improves Autophagic Flux Carloni et al. [49] showed that neuronal autophagy is higher in the neocortex and hippocampus of the ipsilateral hemisphere by 72 h post-HI in P7 rats, raising the possibility that autophagy was either contributing to the evolution of injury or that it was induced as a neuroprotective mechanism.Therefore, in a recent study, we administered SB505124 after perinatal HI and evaluated its effect upon the process of autophagy.We assessed the levels of two key proteins known to mediate autophagy: p62 and LC3.SB505124 increased both the levels of these proteins as well as the number of autophagic granules within the neocortical neurons [50].Additionally, when SB505124 was administered ex vivo, to organotypic slices produced from rat brains 3 days after inducing HI at P6, it enhanced autophagy flux in the injured hemisphere.Furthermore, Fig. 5. Repeated administration of SB505124 improves sensorimotor function after HI injury on P7. a Outline of the experimental paradigm of HI injury and behavioral testing.At 3 days after injury on P7, mouse pups were injected with SB505124 (10 mg/kg) once every 12 h for 5 days.Sensorimotor testing began 21 days following HI injury.Mice were given a pre-training session 24 h before the start of testing.b Body masses for sham injured, HI injured with vehicle, and SB505124-administered HI-injured mouse pups tracked during recovery from HI until the pups were perfused at P30 (n = 8-10 mice per group).c Hemisphere ratio (IL:CL) with representative images of brains.d The modified neurological severity score (mNSS) which evaluated 9 different tasks for a scale of 0-9 (normal score, 0; maximal deficit score, 9).e Average foot slips per run on 1.0 cm wide balance beam.f Sticky paper test measured time (s) to measure adhesive on the contralateral forepaw (n = 5-8 mice per group).g Average foot slips per run on horizontal ladder with the increased difficulty by placing the steps in an irregular fashion.Data represent means ± SEM; *p < 0.05, **p < 0.001 by ANOVA followed by Tukey's post hoc test.Delayed ALK5 Antagonism Protects the Immature Brain from HI administering the membrane-permeable and autophagyinducing peptide TAT-beclin1 [51] to HI-injured rat pups increased autophagic function and preserved hippocampal and thalamic integrity and improved sensorimotor function [52].Here, we sought to identify other mechanisms of ALK5 inhibition and did not evaluate autophagic flux; however, it is very likely that SB505124 is increasing autophagic flux to reduce the extent of damage.
Compromised BBB Function and Tertiary Neurodegeneration Vesicular transport and tight junction complexes regulate the permeability of the vascular endothelial cell monolayer and comprise the functional components of the BBB.Molecules involved include the adherent junction protein, N-cadherin; the tight junction protein, claudin-5; and the water channel, AQP4.TGF-β1 has been shown to alter the neurovascular properties of pericytes [46,53].Indeed, in a study of the consequences of a traumatic brain injury, Patel et al. [54] showed that the oxidative stress caused by the injury activated TGF-β1 and matrix metalloproteinases that lead to disruption of the BBB.Similarly, in another study that used a mouse model of TBI, levels of n-cadherin were reduced in association with the severity of the injury [53].These studies led us to formulate the hypothesis that the tertiary neurodegeneration due to increased TGF-β signaling after HI similarly disrupted the crosstalk between the pericytes and the cerebral endothelium leading to a disturbed BBB, which would lead to progressive brain damage.Supporting that hypothesis, we show that HI reduced levels of 3 key proteins involved in BBB integrity, n-cadherin, aquaporin-4, and claudin-5 at 6 days of recovery from HI and that SB505124 treatment restored or even increased the levels of those proteins.
The strong increase in aquaporin-4 levels was unexpected, but aquaporin-4 is well known to regulate water transport in and out of the brain parenchyma [55].Therefore, the increase in aquaporin-4 expression with SB505123 treatment may have contributed to reduced edema, which in turn may have affected the pressure differences between the ventricles and the parenchyma, as reflected by less ventricular expansion.
In addition to TGF-β itself contributing to the evolution of brain injury, serum albumin leaking into the parenchyma independently activates astrocytic ALK5 to enhance the formation of excitatory synapses, resulting in increased epileptiform activity [56].Intracerebral delivery of the ALK5 inhibitor, SJN2511, prevented albumin-induced seizures in mice.These findings provide yet another mechanism whereby activated TGFβ receptor signaling secondary to BBB compromise and albumin leakage into the brain can induce a hyper-excitatory state to produce brain damage.

Conclusion
Long-term neurological morbidity remains a problem due to the insufficient therapies for neonatal encephalopathy.Therapeutic hypothermia is contraindicated in preterm infants and is not fully protective, thus the survivors often face major lifelong neurodevelopmental disabilities.Clearly, more effective therapeutics are needed.The robust neuroprotection seen in these studies together with the lack of adverse events and the significant improvement in neurological functions support the conclusion that additional studies on the use of SB505124 or similar drugs is warranted for the treatment of perinatal HI in preterm infants.For each item, the animals were awarded either a score of 1 or 0 according to the inability or capacity to perform the test or for the lack or presence of a tested reflex; thus, the higher scores indicate more severe behavioral deficits.

Fig. 4 . 5 (
Fig. 4. Repeated administration of SB505124 preserves white matter integrity after HI injury at P7. Luxol fast blue (LFB) stained sections at 0.2 mm from bregma were analyzed 3 weeks after HI injury at P7. a Ipsilateral white matter integrity was assessed in terms of the area.b-d Representative LFB-stained images taken near the middle cerebral artery (MCA) from sham, vehicle Rx HI, and SB505124 Rx HI (n = 4 animals per group).Data represent means ± SEM; *p < 0.05, **p < 0.001 by ANOVA followed by Tukey's post hoc test.