Memantine improves outcomes after repetitive traumatic brain injury

Abstract

Repetitive mild traumatic brain injury (rmTBI; e.g., sports concussions) is common and results in significant cognitive impairment. Targeted therapies for rmTBI are lacking, though evidence from other injury models indicates that targeting N-methyl-d-aspartate (NMDA) receptor (NMDAR)-mediated glutamatergic toxicity might mitigate rmTBI-induced neurologic deficits. However, there is a paucity of preclinical or clinical data regarding NMDAR antagonist efficacy in the rmTBI setting. To test whether NMDAR antagonist therapy improves outcomes after rmTBI, mice were subjected to rmTBI injury (4 injuries in 4days) and randomized to treatment with the NMDA antagonist memantine or with vehicle. Functional outcomes were assessed by motor, anxiety/impulsivity and mnemonic behavioral tests. At the synaptic level, NMDAR-dependent long-term potentiation (LTP) was assessed in isolated neocortical slices. At the molecular level, the magnitude of gliosis and tau hyper-phosphorylation was tested by Western blot and immunostaining, and NMDAR subunit expression was evaluated by Western blot and polymerase chain reaction (PCR). Compared to vehicle-treated mice, memantine-treated mice had reduced tau phosphorylation at acute time points after injury, and less glial activation and LTP deficit 1 month after injury. Treatment with memantine also corresponded to normal NMDAR expression after rmTBI. No corresponding protection in behavior outcomes was observed. Here we found NMDAR antagonist therapy may improve histopathological and functional outcomes after rmTBI, though without consistent corresponding improvement in behavioral outcomes. These data raise prospects for therapeutic post-concussive NMDAR antagonism, particularly in athletes and warriors, who suffer functional impairment and neurodegenerative sequelae after multiple concussions.

Keywords: Concussion; NMDAR; Repetitive concussion; Traumatic brain injury.

Figure 1. Acute memantine treatment suppresses APP increase after rmTBI

Memantine was given acutely after each injury and APP expression was examined 3 days after last injury. (A) a representative image of APP and β-actin Western blot image. (B). Densitometry was used for semi-quantifying intensity of protein expression. The expression of APP was normalized by βactin expression and further compared to sham group. *compared to sham group, p<0.05; # compared to vehicle treated injury group, p<0.05; n = 8/group.

Figure 2. Acute increase of tau phosphorylation after rmTBI

The cortices were collected from sham, injured vehicle or injured memantine treated mice 3 days after last injury. Phosphorylated tau (T231) and total tau expressions were examined by Western blot. (A) representative Images of Western blots. (B) semi-quantitative results using densitometry. * injury vs. sham, p<0.05; # memantine vs. vehicle, p<0.05. Data are presented as mean ± SEM, n = 8/group

Figure 3. Memantine treatment restores NR1 and NR2B expression 1 month after rmTBI are rescued in injured memantine treated mice compared to vehicle treated mice

(A) representative image of NR2B immunoblotting. (B) quantitatively analyed NR2B protein expression.(C) mRNA of NR2B expression. (D) representative image of NR1 immunoblotting. (E) quantitative expression of NR1 protein and (F) mRNA expression of NR1 analyzed by real time PCR.*p<0.05 compared to sham, #p<0.05 compared to vehicle. Data are presented as means ± SE, n = 8/group.

Figure 4. Treatment with memantine partially restores the LTP deficit after rmTBI

(A) The LTP responses induced by HFS in M1 slices. LTP magnitude (fEPSP slope change 30min after HFS relative to baseline) was attenuated in the vehicle treated mice (Veh: 110.4 ± 0.8% of baseline; n=9, p<0.001) as compared to sham (183.6 ± 1.7% of baseline; n=8, p<0.001). In the memantine (MEM) treated group, this LTP deficit was partially recovered (132.6 ± 2.4% of baseline; n=7, p<0.001). (B) Statistic analysis of the fEPSP slopes changes averaged from the last 10-min’s (45–55min) recording of each group [F(2,30)=786.3, p<0.001 by one-way ANOVA]: ***p<0.001 indicates post hoc test between two means as indicated in the graph. ^###p<0.001 indicates paired t-test between fESPS slope changes and individual baseline (initial 10-min’s recording). n = 8/group

Figure 5. Treatment with memantine attenuates increase of microglial cell after rmTBI

(A) IBA1 staining images in hippocampus with lower (left panel) and higher (right panel) magnifications. (B) IBA1 positive cells in left hippocampus from 3 coronal sections (bragma −1.62, −1.86 and −2.1mm) each mouse were counted under microscope.* p<0.05, vehicle vs. sham, #p<0.05, memantine vs. vehicle. The data are presented as mean/section ± SEM, n= 6/group.

Figure 6. Behavior outcomes

(A) Rotarod task demonstrated deficits of motor balance in injured mice compared compared to sham, n = 28 injured memantine-treated, n= 28 injured vehicle-treated, n = 23 sham,* p<0.05 compared to sham (data are mean± SEM). (B) During elevated plus maze test, injured memantine-treated mice spent less time in closed arm and more time in open arm compared to sham and injured vehicle treated mice, n = 28 injured memantine-treated, n= 28 injured vehicle-treated, n = 24 sham, *p<0.05 compared to sham (data are mean± SEM). (C) In open field test, the injured memantine-treated mice spent less time in outer ring and more time in neutral ring compared to sham and injued vehicle-treated groups, n = 28 injured memantine-treated, n = 28 injured vehicle-treated, n = 24 sham,*p<0.05 compared to sham. No difference of time spent in inner ring (data are mean± SEM). (D) In Morris water maze test, both vehicle and memantine treated injured groups mice had significant deficit of spatial memory, though the effect of injury was worse in injured memantine treated mice, n = 21 injured memantine-treated, n = 21 injured vehicle-treated, n = 18 sham. Continuous variables were compared between injured and sham injured mice and memantine treated versus vehicle treated mice at single time points using analysis of variance (ANOVA) or Kruskal-Wallis for univariate testing as appropriate. To account for repeated measures over time, MWM and rotorod latencies were analyzed by linear regression with clustered, robust standard errors.