Minocycline synergizes with N-acetylcysteine and improves cognition and memory following traumatic brain injury in rats

Abstract

Background: There are no drugs presently available to treat traumatic brain injury (TBI). A variety of single drugs have failed clinical trials suggesting a role for drug combinations. Drug combinations acting synergistically often provide the greatest combination of potency and safety. The drugs examined (minocycline (MINO), N-acetylcysteine (NAC), simvastatin, cyclosporine A, and progesterone) had FDA-approval for uses other than TBI and limited brain injury in experimental TBI models.

Methodology/principal findings: Drugs were dosed one hour after injury using the controlled cortical impact (CCI) TBI model in adult rats. One week later, drugs were tested for efficacy and drug combinations tested for synergy on a hierarchy of behavioral tests that included active place avoidance testing. As monotherapy, only MINO improved acquisition of the massed version of active place avoidance that required memory lasting less than two hours. MINO-treated animals, however, were impaired during the spaced version of the same avoidance task that required 24-hour memory retention. Co-administration of NAC with MINO synergistically improved spaced learning. Examination of brain histology 2 weeks after injury suggested that MINO plus NAC preserved white, but not grey matter, since lesion volume was unaffected, yet myelin loss was attenuated. When dosed 3 hours before injury, MINO plus NAC as single drugs had no effect on interleukin-1 formation; together they synergistically lowered interleukin-1 levels. This effect on interleukin-1 was not observed when the drugs were dosed one hour after injury.

Conclusions/significance: These observations suggest a potentially valuable role for MINO plus NAC to treat TBI.

Figure 1. MINO provided a modest improvement of active place avoidance following moderate CCI.

The total number of entrances into the shock zone was assayed in the 6 trials of active place avoidance training.

Figure 2. MINO plus NAC synergistically improved active place avoidance after massed training.

Panel A, Rats received either CCI or sham-CCI. One hour, one or days later the sham-injured the CCI-injured rats were divided into 4 groups; one group received saline treatment. The remaining CCI-injured rats received either MINO or NAC alone, or the combination of MINO plus NAC. Saline or drug treatments were administered 1 hour, 1 day and 2 days after injury. Rats received active place avoidance training 8 and 9 days after injury. On the 8th and 9th day following CCI, the number of shock zone entrances was measured. Panel B, Representative tracks of rats in the sham-CCI-saline, CCI-saline or CCI-MINO plus NAC groups on the 6^th trial on the first day of active avoidance training. Red lines indicate the boundaries of the shock zone and the red circles indicate the location were a rat received a shock. Panel C, Summary of the number of shock zone entrances over the two days of active place avoidance.

Figure 3. MINO plus NAC synergistically improved active place avoidance during spaced training that requires 24-hour memory.

Panel A, Experimental Design. Rats received moderate CCI or sham-injury. Saline or drug treatments were administered 1 hour, 1 day and 2 days after injury. Beginning 8 days after injury, rats received spaced training of active place avoidance consisting of a 20-minute trial each day for 15 days. Panel B, The number of entrances into the shock zone measured overall avoidance learning. Panel C, Time to 1st entrance into the shock zone measured learning that depended on 24-hour retention. These data suggest that long-term memory was improved by MINO plus NAC but not MINO. MINO plus NAC acted synergistically since the improvement in time to enter the shock zone occurred only with co-administration of both drugs.

Figure 4. MINO plus NAC prevented myelin loss.

Panel A, Schematic of the regions of interest (ROIs) from a coronal section located −3.36 mm from Bregma . The ROIs were: corpus callosum (A), dorsal hippocampal commissure (B), stratum radiatum (C), fimbria (D), internal capsule (E), fornix (F), mammilothalamic tract (G). Panel B, Representative images of corpus callosum and dorsal hippocampal commissure stained with luxol fast blue. B1, Sham-CCI-saline; B2 CCI-saline; B3, CCI-MINO plus NAC. The scale bar corresponds to 100µm.

Figure 5. MINO or MINO plus NAC prevented IL-1β formation when administered before moderate CCI.

Rats were dosed with saline, MINO, NAC or both drugs 3 hours prior to sham- or moderate CCI. The rats were sacrificed one hour after surgery; and IL-1β and tubulin levels were assayed from protein extracts prepared from hippocampus. Panel A, Representative Il-1β and tubulin immunoblot analysis, Panel B, Summary of immunoblot analysis.