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

Abstract

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)β1 is highly elevated following HI and that delivering an antagonist for TGFβ 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.

Keywords: autophagy; cell death; encephalopathy; neuroprotection; premature birth.

Figure 1

SB505124 diminished the number of actively apoptotic cells in the neocortex after hypoxia-ischemia (HI) injury. One week after HI injury at P6, samples of the injured forebrain were analyzed for actively dying cells using in situ end labeling (ISEL). SB505124 or vehicle was administered via osmotic pump beginning at three days after injury and maintained for four days prior to intracardiac perfusion. Thirty µm sections were processed for ISEL. Cells with green nuclei indicate nicked DNA strands. (A) Panels depict representative neocortical cells in the ischemic penumbra of injury in vehicle-treated and SB505124-treated animals. Scale bars in merged image represent 20 µm. (B) Quantitative analysis of the number of ISEL+ cells of the neocortex per mm². n = 4 per group. Data are presented as means ± SEM ** p < 0.001 by Student’s t-test.

Figure 2

SB505124 induced autophagic flux in the injured hemisphere after HI Injury. Three days after HI, 1000 µm thick coronal slices were incubated with SB505124 (SB) or SB505124 + chloroquine (SB+Clq) in DMEM w/20% horse serum (Media Only control, M.O.) for 2 h total in 37 °C, 5% CO₂ to assess the effect of SB505124 on active autophagy. The injured hemisphere was collected and protein extracted for Western blot. (A) Representative blot for LC3, p62, and β-Actin (loading control) from the injured hemisphere. (B) Quantitative analysis of band optical densities for LC3-I, (C) LC3-II, (D) ratio of LC3-II/LC3-I band densities, and (E) band optical densities for p62. n = 4–5 per group. Data are presented as means ± SEM; * p < 0.05, ** p < 0.001 when denoted by bracket; ** p < 0.001 for M.O. vs. SB treated slices; ^## p < 0.001, ^### p < 0.0001 for Clq treated vs. SB+Clq treated slices; ^$ p < 0.05, ^$$ p < 0.001, ^$$$ p < 0.0001 for SB treated vs. SB+Clq treated slices by two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 3

Systemic Tat-Beclin1 administration induced autophagy in the brain up to 48 h after injection. Three days after HI, rat pups were injected i.p. with Tat-Beclin1 (15 mg/kg, TB1) or vehicle (PBS). 48 h after injection, samples of the injured forebrain were prepared for Western blot. (A) Representative blot for LC3, p62, and β-Actin (loading control) from the injured hemisphere. (B) Quantitative analysis of band optical densities for p62 and (C) ratio of LC3-II/LC3-I band densities (Sham n = 5/group; Treatment n = 6/group). Data are presented as means ± SEM; * p < 0.05, ** p < 0.001 as determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 4

Systemically administered Tat-Beclin1 improved sensorimotor outcomes after HI injury. Two weeks after HI injury at P6, behavioral tests were performed to assess sensorimotor function. (A) Outline of the experimental paradigm of HI injury and behavioral testing. Three days after injury on P6, rat pups were injected once with Tat-Beclin1 (15 mg/kg), and again 72 h after the first injection. Sensorimotor testing began 13 days following HI injury. Rats were given a pre-training session 24 h before the start of testing. (B) Body mass of Sham-injured, HI-injured with vehicle and Tat-Beclin1 (TB1) administered rat pups tracked for two weeks after HI injury on P6. (C) Hemispheric ratio (IL:CL) with representative images of brains following extraction. (D) Average slips per run on 2.5 cm-wide balance beam. (E) Average slips per run on inclined 2.5 cm-wide balance beam, (F) Time to drop (s) for hanging bar, (G) mNSS score. n = 9 per group. Data are presented as means ± SEM; * p < 0.05, ** p < 0.001, *** p < 0.0001; ^# p < 0.05 for vehicle-treated vs. Tat-Beclin1 treated rats using one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 5

Systemically administered Tat-Beclin1 reduced hippocampal and thalamic neurodegeneration after HI injury. Cresyl Violet stained sections at +3 mm from Bregma were analyzed two weeks after HI injury at P6. (A) Areas of the hippocampus and thalamus were measured and normalized to contralateral structures. (B) Representative images of structural loss of hippocampal (enclosed by dashed line) and thalamic regions as compared to the contralateral hemisphere n = 3 for Sham group; n = 5 per HI groups. Data are presented as means ± SEM, ** p < 0.001 by one-way ANOVA followed by Tukey’s multiple comparison test.