Preclinical efficacy testing in middle-aged rats: nicotinamide, a novel neuroprotectant, demonstrates diminished preclinical efficacy after controlled cortical impact

Abstract

Age is a consistent predictor of poor outcome following traumatic brain injury (TBI). Although the elderly population has one of the highest rates of TBI-related hospitalization and death, few preclinical studies have attempted to model and treat TBI in the aged population. Recent studies have indicated that nicotinamide (NAM), a soluble B-group vitamin, improved functional recovery in experimental models of TBI in young animals. The purpose of the present study was to examine the preclinical efficacy of NAM in middle-aged rats. Groups of middle-aged (14-month-old) rats were assigned to NAM (500 mg/kg or 50 mg/kg) or saline alone (1 mL/kg) treatment conditions, and received unilateral cortical contusion injuries (CCI) and injections at 1 h and 24 h following injury. The animals were tested on a variety of tasks to assess vestibulomotor (tapered beam) and cognitive performance (reference and working memory in the Morris water maze), and were evaluated for lesion size, blood-brain barrier compromise, astrocytic activation, and edema formation. In summary, the preclinical efficacy of NAM as a treatment following CCI in middle-aged rats differs from that previously documented in younger rats; while treatment with 50 mg/kg NAM appeared to have no effect, the 500-mg/kg dose worsened performance in middle-aged animals. Histological indicators demonstrated more nuanced group differences, indicating that NAM may positively impact some of the cellular cascades following injury, but were not substantial enough to improve functional recovery. These findings emphasize the need to examine potential treatments for TBI utilizing non-standard populations, and may explain why so many treatments have failed in clinical trials.

FIG. 1.

Administration of nicotinamide (NAM) did not improve recovery on the tapered beam walk task in 14-month-old animals. The graph shows plotted mean (±standard error of the mean) percentages of steps that were faults. On days 4, 8, and 10, the 500 mg/kg NAM-treated group faulted significantly more than the saline-treated group. Treatment with 500 mg/kg NAM following injury in 14-month-old animals worsened functional recovery following injury (*p < 0.05), whereas treatment with 50 mg/kg failed to improve recovery.

FIG. 2.

Administration of nicotinamide (NAM) did not improve reference memory performance in the Morris water maze (MWM) in 14-month-old animals. The graph shows plotted mean (±standard error of the mean) distances swam to the platform. On day 16, the 500 mg/kg NAM-treated group performed significantly worse than the sham-injured saline-treated group. Trends in performance appear to indicate that performance on this task is sometimes impaired following NAM administration in injured, 14-month-old animals at the higher dose, and ineffective at the lower dose.

FIG. 3.

Administration of nicotinamide (NAM) did not improve working memory performance in the Morris water maze (MWM). The graph shows plotted mean (±standard error of the mean) distance swam to the platform. On day 19, the 500 mg/kg NAM-treated group performed significantly worse than the sham-injured group. Both the 500 mg/kg NAM-treated and the saline-treated injured groups performed significantly worse than the sham-injured group on day 21. On day 22, both the NAM-treated injured groups performed significantly worse than the sham-injured group, but the saline-treated group did not.

FIG. 4.

Administration of nicotinamide (NAM) did not reduce injury size. Analysis of the percent reduction in injured cortical volume compared to the contralateral cortex is shown. The graph shows plotted mean (±standard error of the mean) percent reductions in cortical volume for each group. Although the injured groups differed significantly from the sham controls, there were no significant differences between the injured groups.

FIG. 5.

Shown are coronal brain sections (40 μm, cresyl violet stain) at the center of the injury cavity (−2.30 mm relative to the bregma) for the different experimental groups. Images shown are representative images from each group: (A) sham, (B) saline, (C) 50 mg/kg NAM, and (D) 500 mg/kg NAM (scale bar = 2.0 mm; NAM, nicotinamide).

FIG. 6.

Administration of nicotinamide (NAM) did not reduce injury size at acute time points. Analysis of the percent reduction in injured cortical volume compared to the contralateral cortex is shown. The graph shows plotted mean (±standard error of the mean) percent reductions in cortical volume for each group. The analysis revealed no statistically significant differences between any of the injured groups in lesion cavity formation.

FIG. 7.

Administration of nicotinamide (NAM) did not significantly reduce reactive gliosis following injury. The graph shows the mean (±standard error of the mean) number of activated astrocytes in cortical areas around the injury cavity. Although administration of NAM reduced glial fibrillary acidic protein (GFAP) expression to some degree compared to saline-treated animals, this effect was not statistically significant.

FIG. 8.

Administration of nicotinamide (NAM) did not reduce BBB breech. The graph shows mean (±standard error of the mean) percent IgG-positive tissue in the whole section. All injured groups showed significantly more staining than the sham-injured group. However, there were no statistically significant differences between the injured groups.

FIG. 9.

Administration of nicotinamide (NAM) did reduce edema following injury in some situations. The graph shows mean (±standard error of the mean) percent water content for the tissue, with higher water content indicating more swelling. At the injury site, both NAM-treated groups had significantly less swelling than the saline-treated group. In the cortex adjacent to the injury, only the saline- and 500-mg/kg NAM-treated groups demonstrated significantly more swelling than the sham-injured group and the 50-mg/kg group. Additionally, the 500 mg/kg NAM-treated group had significantly more swelling than the 50 mg/kg NAM-treated group (*p < 0.05).