TrkB agonist antibody pretreatment enhances neuronal survival and long-term sensory motor function following hypoxic ischemic injury in neonatal rats

Abstract

Perinatal hypoxic ischemia (H-I) causes brain damage and long-term neurological impairments, leading to motor dysfunctions and cerebral palsy. Many studies have demonstrated that the TrkB-ERK1/2 signaling pathway plays a key role in mediating the protective effect of brain-derived neurotrophic factor (BDNF) following perinatal H-I brain injury in experimental animals. In the present study, we explored the neuroprotective effects of the TrkB-specific agonist monoclonal antibody 29D7 on H-I brain injury in neonatal rats. First, we found that intracerebroventricular (icv) administration of 29D7 in normal P7 rats markedly increased the levels of phosphorylated ERK1/2 and phosphorylated AKT in neurons up to 24 h. Second, P7 rats received icv administration of 29D7 and subjected to H-I injury induced by unilateral carotid artery ligation and exposure to hypoxia (8% oxygen). We found that 29D7, to a similar extent to BDNF, significantly inhibited activation of caspase-3, a biochemical hallmark of apoptosis, following H-I injury. Third, we found that this 29D7-mediated neuroprotective action persisted at least up to 5 weeks post-H-I injury as assessed by brain tissue loss, implicating long-term neurotrophic effects rather than an acute delay of cell death. Moreover, the long-term neuroprotective effect of 29D7 was tightly correlated with sensorimotor functional recovery as assessed by a tape-removal test, while 29D7 did not significantly improve rotarod performance. Taken together, these findings demonstrate that pretreatment with the TrkB-selective agonist 29D7 significantly increases neuronal survival and behavioral recovery following neonatal hypoxic-ischemic brain injury.

Figure 1. 29D7 increases phosphorylation of ERK1/2 and AKT in vivo.

P7 rat pups received an icv injection of control IgG or 29D7 (0.3 nmol in 5 µl PBS) and the cortical tissues were dissected at various time points as indicated. Following preparation of tissue lysates, proteins (30 µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed with antibodies specific to phosphorylated ERK1/2 (pERK1/2) and total ERK1/2, and phosphorylated AKT (pAKT) and total AKT proteins. Signal intensity was quantified by densitometry and normalized to corresponding total proteins. Since IgG treatment had no effect on the levels of pERK1/2 and pAKT (P>0.05) for up to 24 h, all time points of the IgG-treated groups were pooled. Data represent mean ± SEM (n = 4–5). *P<0.05 compared with the IgG-treated group by ANOVA followed by Dunnett’s multiple comparison (A–B). C. Levels of phosphorylated ERK1/2 and phosphorylated AKT were compared in male vs. female mice 2–6 h after icv injection of 29D7, 0.3 nmol (n = 5). N.S.: no significance between male and female mice assessed by t-test (P>0.05).

Figure 2. 29D7 induces phosphorylation of ERK1/2 in neurons.

P7 rats received an icv injection of either IgG or 0.3(n = 5 per group). Coronally sectioned brain sections were immunofluorescently double-labeled with anti-phospho-ERK1/2 (pERK1/2) antibody (red) and a neuron-specific marker, NeuN (green), followed by fluorescent microscopy. Note that pERK1/2 signals are co-localized with NeuN staining (indicated by yellow signals) in the cortex ipsilateral to icv injection. Scale bar: 50 µm.

Figure 3. 29D7 blocks caspase-3 activation following H-I brain injury.

P7 rats underwent left carotid ligation followed by hypoxic injury for 2.5-four hours later, brain tissues were dissected from the hippocampus (A, C) and the cortex (B, D). A, B. Tissues were lysed and caspase-3 activity was determined by Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (DEVD-AMC) cleavage assay. Data indicate mean ± SEM. * p<0.05 compared with contralateral hemisphere; # p<0.05 compared with a IgG-treated ipsilateral hemisphere, as analyzed by ANOVA followed by Dunnett’s comparison method. C, D. Tissue proteins (30 µg/lane) were separated by SDS-PAGE and subjected to immunoblotting with antibodies specific to poly(ADP-ribose) polymerase (PARP), α-spectrin, and β-actin. Data shown are representative of 6 independent experiments.

Figure 4. 29D7 exerts long-term neuroprotection against H-I brain injury.

P7 rats underwent left carotid ligation and hypoxia for 2.5(0.1 nmol or 0.3 nmol) prior to hypoxia. Seven days later, brains were removed, coronally sectioned, and stained with cresyl violet. A. Representative photographs illustrating hemispheric tissue loss after H-I injury. B. Area tissue loss from the striatum, hippocampus, and cortex was determined by comparing the area of surviving tissue with unlesioned (right) hemisphere. Data indicate mean ± SEM. *P<0.05 compared to the IgG-treated group. C. Animals used in behavioral studies in Fig. 5 were subjected to tissue loss analysis at P42. *P<0.05 compared to the sham-operated group; #P<0.05 compared to a control IgG-treated, H-I group.

Figure 5. 29D7 does not affect body temperature.

P7 rats underwent left common carotid ligation or sham surgery, followed by hypoxia for 2.5-I injury were treated with an icv administration of control IgG or 29D7 immediately before hypoxia. Body temperature of the pups was measured at various time points indicated using a digital infrared thermometer. Data indicate mean ± SEM. No significance between groups assessed by one way ANOVA (P>0.05).

Figure 6. 29D7 improves functional outcome following H-I injury.

P7 rats underwent sham operation (Sham) or H-I brain injury by left carotid ligation and subsequent exposure to hypoxia for 2.5 h. H-I animals received an icv administration of 0.3 nmol control IgG or 29D7, 0.3 nmol prior to hypoxia. A. Sensorimotor function was assessed by a tape-removal test at P28, P35, and P42. Data represent mean ± SEM. *P<0.05 compared with sham; #P<0.05 compared with IgG treated group, analyzed by ANOVA and Dunnett’s multiple comparison test. B. Correlation between % cortical tissue loss and functional performances. Functional performance of individual animals in A was plotted against the percentage of cortical tissue loss, as exemplified in Figure 4C. C. Motor coordination function was assessed by a rotarod test 3 weeks after H-I injury. P>0.05 between groups.