D-cycloserine improves functional outcome after traumatic brain injury with wide therapeutic window

Abstract

It has been long thought that hyperactivation of N-methyl-D-aspartate (NMDA) receptors underlies neurological decline after traumatic brain injury. However, all clinical trials with NMDA receptor antagonists failed. Since NMDA receptors are down-regulated from 4h to 2weeks after brain injury, activation at 24h, rather than inhibition, of these receptors, was previously shown to be beneficial in mice. Here, we tested the therapeutic window, dose regimen and mechanism of action of the NMDA receptor partial agonist D-cycloserine (DCS) in traumatic brain injury. Male mice were subjected to trauma using a weight-drop model, and administered 10mg/kg (i.p.) DCS or vehicle once (8, 16, 24, or 72h) twice (24 and 48h) or three times (24, 48 and 72h). Functional recovery was assessed for up to 60days, using a Neurological Severity Score that measures neurobehavioral parameters. In all groups in which treatment was begun at 24 or 72h neurobehavioral function was significantly better than in the vehicle-treated groups. Additional doses, on days 2 and 3 did not further improve recovery. Mice treated at 8h or 16h post injury did not differ from the vehicle-treated controls. Co-administration of the NMDA receptor antagonist MK-801 completely blocked the protective effect of DCS given at 24h. Infarct volume measured by 2,3,5-triphenyltetrazolium chloride staining at 48h or by cresyl violet at 28days was not affected by DCS treatment. Since DCS is used clinically for other indications, the present study offers a novel approach for treating human traumatic brain injury with a therapeutic window of at least 24h.

Figure 1

Effect of DCS dose on functional recovery after head injury. The Neurological Severity Score (NSS) tests reflexes, alertness, coordination and motor abilities. The extent of recovery is calculated as the difference between NSS at 1h and that at any other time: ΔNSS(t) = NSS (1h) – NSS (t). A. DCS was given at 8 or 16h. B. DCS was given 24 or 72h after injury. Vehicle was given at the same intervals. *P<0.05 DCS given at 24h or 72h vs saline, given at the same time (Mann-Whitney non-parametric test)

Figure 2

The effect of multiple doses of DCS on functional recovery. DCS was given twice (DCS-2), at 24 and 48h, or three times (DCS-3, 24, 48 and 72h) after closed head injury. *P<0.05 and **P<0.01, both treatment groups vs saline (Mann-Whitney non-parametric test).

Figure 3

The effect of DCS on infarct volume, assessed 24 h after drug administration (48 h after closed head injury). A. Brain section immersed in 2% TTC in PBS and photographed using a Stemi SV11 Stereoscope (Zeiss, Germany) and digital photocamera Coolpix E990 (Nikon, Japan). Black arrow indicates infracted area. B. Area of hemispheres ipsilateral and contralateral to the trauma was measured and percent hemispheric infarct volume was quantified using the Scion Image-Release Beta 4.0.2 program.

Figure 4

Histology at impact level 28 days after injury. Cryostat sections were stained with cresyl violet. Note cavitation on left (ipsilateral side) and ventricular enlargement on right (contralateral) side (A). The area of the hemispheres ipsilateral and contralateral to the trauma was measured using Image J software and the percent difference between the two was calculated to estimate long-term tissue loss (B).

Figure 5

MK-801 reversal of the DCS effect on functional recovery. Mice were treated 24h after closed head injury with vehicle, MK-801, DCS or both drugs together. NSS was followed for 3 weeks after injury. *P<0.05 vs DCS+MK-801 group.