Memantine inhibits cortical spreading depolarization and improves neurovascular function following repetitive traumatic brain injury

Abstract

Cortical spreading depolarization (CSD) is a promising target for neuroprotective therapy in traumatic brain injury (TBI). We explored the effect of NMDA receptor antagonism on electrically triggered CSDs in healthy and brain-injured animals. Rats received either one moderate or four daily repetitive mild closed head impacts (rmTBI). Ninety-three animals underwent craniectomy with electrocorticographic (ECoG) and local blood flow monitoring. In brain-injured animals, ketamine or memantine inhibited CSDs in 44 to 88% and 50 to 67% of cases, respectively. Near-DC/AC-ECoG amplitude was reduced by 44 to 75% and 52 to 67%, and duration by 39 to 87% and 61 to 78%, respectively. Daily memantine significantly reduced spreading depression and oligemia following CSD. Animals (N = 31) were randomized to either memantine (10 mg/kg) or saline with daily neurobehavioral testing. Memantine-treated animals had higher neurological scores. We demonstrate that memantine improved neurovascular function following CSD in sham and brain-injured animals. Memantine also prevented neurological decline in a blinded, preclinical randomized rmTBI trial.

Fig. 1.. Experimental study design.

(A) Closed head injury models of TBI. Nine-week-old, male Sprague-Dawley rats received either mild (500 g mass, 85 cm height) or moderate (450 g mass, 1.02 m height) head impacts via a modified Marmarou weight drop. rmTBI involved one mild TBI per day for up to four consecutive days. A subgroup of rmTBI animals received memantine (10 mg/kg, intraperitoneally) after the first and before the second, third, and fourth impacts (rmTBI-DM). Sham animals received brief isoflurane anesthetic without TBI. (B) Microsurgical model to investigate effects of NMDAR antagonism on in vivo ECoG and vascular parameters following CSDs. Sham and brain-injured animals underwent cranial window surgery 24 hours following final impact or brief isoflurane anesthetic (sham). Experiments were stratified by impact severity, drug, and route of delivery. Vehicle (2.5 cm³/kg normal saline, intraperitoneally), ketamine (100 μM, topically applied to the exposed cortex), ketamine (25 mg/kg, intraperitoneally), and memantine (10 mg/kg, intraperitoneally) were tested in vivo. Treatment group and sample sizes are described in Results and table S1. (C) Blinded, preclinical randomized trial of NMDAR antagonism for the treatment of rmTBI. Following a single-arm pilot trial of rmTBI, in which animals (n = 13) received memantine (10 mg/kg, intraperitoneally), 31 animals were blindly randomized to memantine (10 mg/kg, intraperitoneally) or saline (2.5 cm³/kg, intraperitoneally). The primary outcome was the proportion of animals that scored ≥6 on the NSS composite (of 12 points).

Fig. 2.. NMDAR antagonism inhibits CSD and reduces CSD amplitude and duration following repeated electrical stimulation of the cortex in sham animals.

(A) Exemplary vascular and ECoG changes during CSD triggered via electrical stimulation of the cortex. Laser Doppler flowmetry was used to record changes in CBF following stimulation. A characteristic negative shift in near-DC/AC-ECoG was observed in cases where CSD occurrence was not inhibited. CSD-induced depression of brain activity is demonstrated via AC-ECoG and integral of AC-ECoG power tracings. (B to D) In each experiment, an initial cortical stimulus was delivered before study drug delivery, serving as an internal control, and demonstrating the ability to elicit a CSD. Heat maps demonstrate the effect of intraperitoneal (IP) delivery of vehicle saline (Veh; 2.5 mg/kg; n = 7), ketamine (Ket; 25 mg/kg; n = 6), or memantine (Mem; 10 mg/kg; n = 9) on CSD-related parameters including occurrence (i.e., percentage of stimuli resulting in CSD), duration, and amplitude (both in relative units) using near-DC/AC ECoG recordings. Repeated cortical stimuli were delivered 45, 60, 75, and 90 min following drug delivery. Topical (top) ketamine (n = 5) was applied to the cortex as 100 μM, with aCSF solvent dilutant. Detailed data pertaining and statistical significance values may be found in table S1.

Fig. 3.. NMDAR antagonism inhibits CSD occurrence and reduces CSD amplitude and duration following repeated electrical stimulation of the cortex in animals following modTBI or rmTBI.

A subset of animals received memantine once per day for four consecutive days while receiving rmTBI (i.e., rmTBI-DM). (A to C) Heat maps demonstrate the effect of intraperitoneal (IP) delivery of vehicle saline (Veh; 2.5 mg/kg; modTBI, n = 5 to 6; rmTBI, n = 6; rmTBI-DM, n = 4 to 6), ketamine (Ket; 25 mg/kg; modTBI, n = 5 to 6), or memantine (Mem; 10 mg/kg; modTBI, n = 5 to 6; rmTBI, n = 5; rmTBI-DM, n = 11) on CSD-related parameters, including occurrence (i.e., percentage of stimuli resulting in CSD), duration, and amplitude (both in relative units) as measured using near-DC/AC-ECoG recordings. The topical (top) (ketamine; 100 μM; modTBI; n = 4) drug delivery route was also tested. (D) Memantine (10 mg/kg, intraperitoneally) reduced the rate of occurrence of CSDs before and after intraoperative drug delivery in each respective TBI model. P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Detailed data pertaining and statistical significance values may be found in table S2.

Fig. 4.. Memantine mitigates brain activity depression during CSD in rmTBI-DM animals.

(A) Normalized AC-ECoG power integral tracings demonstrate the effect of intraperitoneal injection of memantine (10 mg/kg) on brain activity during the 300-s period following each respective cortical stimulation (Stim) before (i.e., stimulations 1 to 2) and after (i.e., stimulations 3 to 5) drug delivery. (B) The nadir (n = 16) was identified as the lowest value of the power integral recording during the 5-min period after stimulation. (C) The rate of change (n = 16) was calculated as the slope of power integral decay from the normalized baseline to its nadir. (D) Area under the curve (AUC) (n = 12) was measured for each stimulation between 150 and 300 s and expressed as a percentage of AUC during a 150-s period preceding the stimulation (baseline). Means and SDs are displayed on bar graphs. P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 5.. Daily memantine use has a lasting effect and reduces CSD-induced spreading depression of brain activity following rmTBI.

(A) Normalized AC-ECoG power integral tracings demonstrate the degree of brain activity depression during the 300-s period following an initial electrical cortical stimulus (stim), before drug delivery, for each respective TBI model (modTBI, rmTBI, and rmTBI-DM). (B) The nadir was identified as the lowest value of the power integral recording during a 5-min period after stimulation. (C) Rate of change represents the slope of power integral decay from the normalized baseline to the respective nadir. (D) Power integral AUCs were measured for each animal group between 150 and 300 s and are expressed as a percentage of the AUC during a 150-s period preceding the stimulation (normalized baseline). Means and SDs are displayed on bar graphs. N values are displayed on the figure. P > 0.05; *P ≤ 0.05; **P ≤ 0.01.

Fig. 6.. Daily memantine use improves local cortical blood flow and reduces the degree of pial arterial vasoconstriction following CSD in rmTBI.

(A) Laser Doppler flowmetry recordings depict hyperemic and oligemic phases of the CBF response to CSD. AUC measurements for the hyperemic (B) and oligemic (C) CBF phases were measured between 30 to 120 s and 150 to 300 s after stimulation, respectively. AUCs for the hyperemic and oligemic phases are expressed as a percentage of the AUC during a 150-s period preceding the stimulation (normalized baseline). (D) Cortical pial artery diameter was measured using direct intravital microscopy. AUCs for vasodilation (E) and vasoconstriction (F) were calculated as an integral of the time-dependent changes in 30 to 120 s and 150 to 300 s after stimulation, respectively, and are represented as a percentage of the AUC during a 150-s period preceding the stimulation (normalized baseline). Means and SDs are displayed on bar graphs. N values are depicted on the figure. P > 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 7.. Memantine improves neurological outcomes following rmTBI.

(A) Pilot trial: mean NSS scores are depicted for animals that received memantine (10 mg/kg, intraperitoneally ) once daily for four consecutive days with rmTBI (i.e., rmTBI-DM; n = 13). (B and C) Preclinical RCT: A blinded RCT of rmTBI-DM (n = 15) versus daily saline (2.5 mg/kg, intraperitoneally; n = 16) was conducted, and individual NSS (B) and mean aggregate scores (C) are demonstrated. (D) NSS subcomponent scores are shown for the aggregate treatment arms (rmTBI, left panel; rmTBI-DM, right panel). (E) Individual NSS values were compared for animals enrolled in the pilot study, the RCT, as well as an additional historical cohort of animals that underwent rmTBI. P > 0.05; **P ≤ 0.01; ***P ≤ 0.001. Means are presented with SEs.