Neuroprotective Effects of Acetyl-L-Carnitine on Neonatal Hypoxia-Ischemia Induced Brain Injury in Rats
Shiyu Tang1,2, Jaylyn Waddell3, Mary C. Mckenna3, Su Xu1,2, Prashant Raghavan1, and Gullapalli P. Rao1,2

1Diagnostic Radiology & Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States, 2Core for Translational Research in Imaging, University of Maryland School of Medicine, Baltimore, MD, United States, 3Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States


Cerebral hypoxia ischemia (HI) is a primary cause of perinatal brain injury. Infants surviving HI face risk of abnormal neurodevelopment, motor and intellectual disability. Acetyl-L-carnitine (ALCAR) is neuroprotective while its efficacy in perinatal HI treatment has yet to be shown. To test the neuroprotective efficacy of ALCAR and potential sex differences, we performed in vivo MRI and behavioral tests in a rat model of neonatal HI. Results revealed that ALCAR is neuroprotective in both morphological and behavioral outcomes. Males exhibit more consistent HI-induced functional impairments. ALCAR was effective in ameliorating functional impairments in both males and females.


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 disability1,2. 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.

Materials and Methods

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 ( DWI data were processed using 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.


This study was supported by NIH grant 5P01 HD016596.


1.Robertson CM, Finer NN, Grace MG. School performance of survivors of neonatal encephalopathy associated with birth asphyxia at term. J Pediatr. 1989;114:753-760.

2. Dixon RM, Bradley KM, Budge MM et al. Longitudinal quantitative proton magnetic resonance spectroscopy of the hippocampus in Alzheimer's disease. Brain 2002; 125(Pt 10):2332-2341.

3. Jones LL, McDonald DA, Borum PR et al. Acylcarnitines: role in brain. Prog. Lipid Res. 2010; 49:61-75

4. Rice JE III, Vannucci RC, Brierley JB et al. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann.Neurol. 1981; 9:131-141.

5. Waddell J, Hanscom M, Shalon Edwards N et al. Exp. Sex differences in cell genesis, hippocampal volume and behavioral outcomes in a rat model of neonatal HI. Neurol. 2015; In Press.


Table 1. Animal grouping information for MRI study and behavioral tests.

Figure 1. A) whole brain, B) ipsilateral hemisphere, C) contralateral hemisphere volume, and D) percentages of lesion to whole brain volume were analyzed with one-way ANOVA and post-hoc analysis (Tukey) within each time post-HI. *=p<0.05, **=p<0.01, ***=p<0.001 HI vs. Sham; #=p<0.05, ##=p<0.01, ###=p<0.001 HI+ALCAR vs. Sham; †=p<0.05 HI vs. HI+ALCAR.

Figure 2. The cortex MD ratio (ipsilateral cortex MD over contralateral cortex MD) were compared between groups with one-way ANOVA and post hoc analysis (Tukey) within each time post-HI.**=p<0.01 HI vs. Sham. Inset: illustrates the ROIs for MD measurement (green: ipsilateral cortex; pink: contralateral cortex)

Figure 3. A) Righting reflexes (72hr post-HI). Only males exhibited a deficit which was reversed by ALCAR. B) Negative geotaxis (7d post-HI). Both males and females showed impairments which was reversed by ALCAR. C) Wire hanging task (PD26). Only males were impaired and improved by ALCAR. *=p<0.05 HI vs. sham.

Figure 4. Novel object recognition testing was conducted between PD32-35. Malesexhibited poor retention at a short (1hr) and a long (24hr) retention delay and this was significantly improved by ALCAR. Females were not significantly impaired. *=p<0.05 HI vs. Sham.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)