Hypoxia ischemia (HI) is a primary cause of perinatal brain injury, occurring at a rate of 1 to 6 per 1000 near-term and term infants in the U.S. Infants surviving perinatal HI face increased risk of abnormal neurodevelopmental outcome, motor and intellectual disability^1,2. Acetyl-L-carnitine (ALCAR) is a dietary supplement which has neuroprotective effects³. However, efficacy of ALCAR therapy for neonatal HI treatment has yet to be shown. We evaluated the longitudinal brain development following ALCAR treatment in a rat model of moderate neonatal HI. Efficacy of ALCAR neuroprotection and sex effects were determined using in vivo magnetic resonance imaging (MRI) and behavioral assessments in separate experiments.

We used a modified Rice-Vanucci model⁴ of HI injury. Animal groups are shown in Table 1. HI was induced at postnatal day (PD) 7 by ligation of the right carotid artery followed by 75 min hypoxia (8% O₂; 92% N₂). Pups recovered at 37°C in room air for 2hr prior to return to the dam. Pups in sham groups were subjected to identical surgery procedure without artery ligation and anesthetized for the same duration as HI pups. ALCAR (200 mg/kg in saline) or saline was injected subcutaneously at 0, 4 and 24hr post-HI.

In vivo MRI was performed on a BrukerBioSpec 7T MR scanner at 24hr, 72hr, 7d and 28d post-HI using isoflurane anesthesia. An MR compatible small-animal monitoring and gating system was used to monitor respiration and body temperature which was kept at 36-37°C using a water circulation pad. A two-dimensional rapid acquisition with relaxation enhancement (RARE) sequence in coronal view (TR/TE = 5500/56.82 ms, FOV = 25 ⨉ 25 mm², slice thickness = 0.5 mm, in-plane resolution = 0.1 × 0.1mm², slice number = 20) was used for T2-weighted imaging acquisition. Diffusion weighted imaging (DWI) was acquired on all three orthogonal axis with b values equal to 350, 700 and 1050 s/mm² along with a non-DWI (b₀ = 0 s/mm²) (TR/TE = 4500/26.91 ms, slice thickness = 1 mm, in-plane resolution = 0.208 × 0.208 mm², slice number = 10).

Animals were tested in a series of behavioral assays (i.e., reflex tests, wire suspension and novel object recognition) as described in recent publication⁵.

Volumetric measurements were conducted using MIPAV (http://mipav.cit.nih.gov/). DWI data were processed using FSLview (http://fsl.fmrib.ox.ac.uk/fsl/fslview/). Diffusivity was calculated in a region of interest (ROI) in the neocortex. Statistical analysis was conducted in SPSS (Ver.19.0, SPSS Inc., Chicago, IL, USA). For MRI experiments, repeated-measures ANOVA was used to test the group ⨉ time post-HI interaction (between-subject factor: group; within-subject variables: time post-HI). One-way ANOVA was used to determine group effect at specific times post-HI when a significant interaction and group effect was detected at p<0.05 level in repeated-measures ANOVA. Post-hoc analysis was performed with Tukey’s test. Behavioral tests were analyzed using ANOVA with treatment (saline/ALCAR) and surgery (sham/HI) as factors. Males and females were analyzed separately to investigate potential sex differences in behavioral study.

Overall effects of treatment and time post-HI ⨉ group interaction were detected in both lesion volume and overall brain volume. Both HI and HI+ALCAR groups had smaller whole brain and ipsilateral hemisphere volumes compared to shams. Contralateral hemisphere was smaller at 28d post-HI only in HI group (Fig 1 A, B, C). ALCAR treatment attenuated lesion development and prevented the increase of lesion volume at 72hr post-HI (Fig 1 D).

Post-hoc analysis showed smaller cortex mean diffusivity (MD) ratio (MD ratio = ipsilateral MD/contralateral MD) in only HI group up to 24hr post-HI (Fig 2). MD ratio increased after 72hr in HI group suggesting delayed cell lysis and edema. ALCAR prevented delayed brain damage in HI+ALCAR group.

ALCAR improved behavioral outcomes in both males and females after HI. ALCAR treatment normalized righting reflex and negative geotaxis, and improved performance in a wire suspension test (Fig 3). Males were more consistently impaired after HI. Only males exhibited a memory deficit in an object recognition test which was reversed by ALCAR (Fig 4).

ALCAR was neuroprotective in both morphological and behavioral outcomes. Males had more consistent functional impairments after HI. ALCAR was effective in ameliorating functional impairments in both males and females.