Evaluation of Altered Glutamatergic Activity in a Piglet Model of Hypoxic-Ischemic Brain Damage Using 1H-MRS

Abstract

Methods: Twenty-five newborn piglets were selected and then randomly assigned to the control group (n = 5) and the model group (n = 20) subjected to HI. HI was induced by blocking bilateral carotid blood flow under simultaneous inhalation of a 6% oxygen mixture. ¹H-MRS data were acquired from the basal ganglia at the following time points after HI: 6, 12, 24, and 72 h. Changes in protein levels of EAAT2 and GluR2 were determined by immunohistochemical analysis. Correlations among metabolite concentrations, metabolite ratios, and the protein levels of EAAT2 and GluR2 were investigated.

Results: The Glu level sharply increased after HI, reached a transient low level of depletion that approached the normal level in the control group, and subsequently increased again. Negative correlations were found between concentrations of Glu and EAAT2 protein levels (R _s = -0.662, P < 0.001) and between the Glu/creatine (Cr) ratio and EAAT2 protein level (R _s = -0.664, P < 0.001). Moreover, changes in GluR2 protein level were significantly and negatively correlated with those in Glu level (the absolute Glu concentration, R _s = -0.797, P < 0.001; Glu/Cr, R _s = -0.567, P = 0.003).

Conclusions: Changes in Glu level measured by ¹H-MRS were inversely correlated with those in EAAT2 and GluR2 protein levels following HI, and the results demonstrated that ¹H-MRS can reflect the early changes of glutamatergic activity in vivo.

Figure 1

Typical images of hematoxylin and eosin staining of the piglet basal ganglia (400x magnification). Scale bar = 50 μm. (a) In the control group, piglet nerve cells were regularly arranged with normal morphology. (b, c) At the later stage following HI, piglet nerve cells were significantly swollen, the cells were slightly stained, the intracellular space was widened, and karyolysis (arrow) was observed.

Figure 2

Changes in GFAP expression in the basal ganglia of piglets (400x magnification). Scale bar = 50 μm. (a–d) Representative figures of GFAP IHC staining in the control group and HI insult 12 h, 24 h, and 72 h subgroups. (e) Changes in the average OD of GFAP protein level. Compared with the control group, the expression levels of the GFAP were significantly increased in the HI group. GFAP: glial fibrillary acidic protein; IHC: immunohistochemical; OD: optical density. Error bars represent the standard deviation values (n = 5/group). ^∗P < 0.05 compared with the control group.

Figure 3

EAAT2 protein levels in the basal ganglia before (control) and after HI injury (400x magnification). Scale bar = 50 μm. (a–d) Representative figures of EAAT2 IHC staining in the control group and in 6 h, 12 h, and 72 h HI subgroups. (e) Changes in the average OD of EAAT2 protein. During early HI, EAAT2 protein level slightly decreased compared with the control; subsequently, it tended to increase and then decrease over time after HI injury. EAAT2: excitatory amino acid transporter 2; IHC: immunohistochemical; OD: optical density. Error bars represent the standard deviation values (n = 5/group). ^∗P < 0.05 compared with the control group.

Figure 4

AMPAR subunit GluR2 protein levels in the basal ganglia before (control) and after HI injury (400x magnification). Scale bar = 50 μm. (a) High GluR2 protein levels in the basal ganglia in the control group. (b) At 72 h after HI injury, the GluR2 protein level was markedly reduced. (c) Changes in the average OD of GluR2 protein level visualized with IHC staining. GluR2 protein level declined in piglets subjected to HI injury compared with the control. (d) GluR2 protein level was negatively correlated with the severity of pathological lesions; i.e., the more severe the brain injury, the more obvious the downregulation of GluR2 protein level. AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid receptor; IHC: immunohistochemical; OD: optical density. Error bars represent the standard deviation values (n = 5/group). ^∗P < 0.05 compared with the control group. The Spearman rank correlation coefficient was presented as R_s.

Figure 5

Representative ¹H-MRS images of the basal ganglia of piglets at different time points after HI and changes in metabolite absolute concentrations and ratios. (a) Axial T2-weighted MR image obtained from a control piglet; the blue box indicates the voxel location. (b) The following metabolite peaks were observed: Glu, Gln, Glx, NAA, Cho, and Cr. (c, d) Changes in metabolite absolute concentrations and metabolite ratios in the control and HI-induced groups at different time points are shown. Glu: glutamate; Gln: glutamine; Glx: glutamate/glutamine complex; NAA: N-acetylaspartate; Cho: choline; Cr: creatine. Error bars represent the standard deviation values (n = 5/group). ^∗P < 0.05 compared with the control group.

Figure 6

Scatter plot of correlations between the expression levels of EAAT2 or AMPAR subunit GluR2 protein levels and the Glu or Glx metabolite levels in the basal ganglia of piglets. EAAT2: excitatory amino acid transporter 2; AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid receptor; OD: optical density; Glu: glutamate; Glx: glutamate/glutamine complex. The Spearman rank correlation coefficient was presented as R_s.