A Combination Therapy of Nicotinamide and Progesterone Improves Functional Recovery following Traumatic Brain Injury

Abstract

Neuroprotection, recovery of function, and gene expression were evaluated in an animal model of traumatic brain injury (TBI) after a combination treatment of nicotinamide (NAM) and progesterone (Prog). Animals received a cortical contusion injury over the sensorimotor cortex, and were treated with either Vehicle, NAM, Prog, or a NAM/Prog combination for 72 h and compared with a craniotomy only (Sham) group. Animals were assessed in a battery of behavioral, sensory, and both fine and gross motor tasks, and given histological assessments at 24 h post-injury to determine lesion cavity size, degenerating neurons, and reactive astrocytes. Microarray-based transcriptional profiling was used to determine treatment-specific changes on gene expression. Our results confirm the beneficial effects of treatment with either NAM or Prog, demonstrating significant improvements in recovery of function and a reduction in lesion cavitation, degenerating neurons, and reactive astrocytes 24 h post-injury. The combination treatment of NAM and Prog led to a significant improvement in both neuroprotection at 24 h post-injury and recovery of function in sensorimotor related tasks when compared with individual treatments. The NAM/Prog-treated group was the only treatment group to show a significant reduction of cortical loss 24 h post-injury. The combination appears to affect inflammatory and immune processes, reducing expression of a significant number of genes in both pathways. Further preclinical trials using NAM and Prog as a combination treatment should be conducted to identify the window of opportunity, determine the optimal duration of treatment, and evaluate the combination in other pre-clinical models of TBI.

Keywords: controlled cortical impact; nicotinamide; polytherapy; progesterone; recovery of function; traumatic brain injury.

FIG. 1.

Functional recovery assessments. (A) Treatment improves sensorimotor function evidenced by shorter latencies (±standard error of the mean [SEM]) to remove the adhesive from the impaired forelimb in the tactile adhesive removal task. All groups were significantly different (p<0.05) than the Vehicle group. The nicotinamide (NAM)/progesterone (Prog)-treated group was significantly different than both individual treatment groups and not significantly different than the Sham group. (B) Treatment improves fine motor performance indicated by the percent (±SEM) usage bias for the unimpaired forelimb in the forelimb asymmetry task using the following formula: Contralateral contacts/(Contralateral contacts+ipsilateral contacts) ×100. All groups were significantly different (p<0.05) than the Vehicle group. The NAM/Prog-treated group was significantly different than the Prog-treated group. (C) Treatment improves fine motor performance evidenced by a reduction in fault scores for the impaired forelimb (±SEM) in the locomotor placing task using the following formula: (Contralateral faults – ipsilateral faults)/lines crossed. All groups were significantly different (p<0.05) than the Vehicle group. The NAM/Prog-treated group was significantly different than both of the individual treatment groups.

FIG. 2.

Functional recovery assessments: main effect graphs. Treatment improves sensorimotor function evidenced by shorter latencies (±standard error of the mean [SEM]) to remove the adhesive from the impaired forelimb in the tactile adhesive removal task as shown collapsed across all days. All groups were significantly different (*p<0.05) than the Vehicle group. The nicotinamide (NAM)/ progesterone (Prog)-treated group was significantly different than both individual treatment groups and not significantly different than the Sham group. (B) Treatment improves fine motor performance indicated by the percent (±SEM) usage bias in the forelimb asymmetry task as shown collapsed across days. All groups were significantly different (*p<0.05) than the Vehicle group. The NAM/Prog-treated group was significantly different than the Prog-treated group. (C) Treatment improves fine motor performance as evidenced by a reduction in fault scores collapsed across days. All groups were significantly different (*p<0.05) than the Vehicle group. The NAM/Prog-treated group was significantly different than both of the individual treatment groups.

FIG. 3.

Lesion analysis. (A) Representative images of cresyl violet–stained (24 h post- cortical contusion injury [CCI]) tissue throughout the injury coordinates: +1.7 mm, +0.9 mm, +0.1 mm, and −0.7 mm, coordinates (anterior/posterior) relative to bregma; scale bar=3.0 mm. (B) Representative images of cresyl violet–stained (24 h post-CCI) tissue throughout the same coordinates. (C) Treatment significantly decreases the percent reduction of cortical volume (±standard error of the mean) between the ipsilateral and contralateral sides to the injury following 24 h and 29 d post-injury using the following formula: 1 - (ipsilateral/contralateral) ×100). At 24 h post-injury, the Sham-treated and the nicotinamide (NAM)/ progesterone (Prog)-treated groups were significantly different (p<0.05) than the Vehicle-treated group. At 29 d post-injury, all groups were significantly different (p<0.05) than the Vehicle group.

FIG. 4.

Pathophysiological analyses. (A) Photomicrograph representing Fluoro-Jade (FJ)+and glial fibrillary acidic protein (GFAP)+stained cell bodies for each treatment group 24 h post-injury; scale bar=100 μm. (B) Treatment reduces the number (±standard error of the mean [SEM]) of FJ+degenerating neurons in the cortices both ipsilateral and contralateral to the injury. In the ipsilateral hemisphere, all groups were significantly different (p<0.05) than the Vehicle group. The nicotinamide (NAM)/progesterone (Prog)-treated group was significantly different than both of the individual treatment groups. There were no significant differences in the contralateral hemisphere. (C) Treatment reduces the number (±SEM) of GFAP+reactive astrocytes in the cortices both ipsilateral and contralateral to the injury. In the ipsilateral hemisphere, all groups were significantly different (p<0.05) than the Vehicle group. The NAM/Prog-treated group was significantly different than both of the individual treatment groups. There were no significant differences in the contralateral hemisphere.

FIG. 5.

The Venn diagrams show the number of genes whose expression was up- or down-regulated more than 1.5-fold (p<0.05) in the progesterone (Prog)/Vehicle, nicotinamide (NAM)/Vehicle, and Prog-NAM/Vehicle contrasts at the 24 h, 72 h, and 7 d time-points. Venn diagrams were generated with the Bioconductor limma package.

FIG. 6.

TaqMan based reverse transcription polymerase chain reaction (RT-PCR) validation of the microarray data for the selected genes: Ccl2 (chemokine (C-C motif) ligand 2), Ccl3 (chemokine (C-C motif) ligand 3), Ccr1 (chcmokine (C-C motif) receptor 1), Clec4e (C-type lectin domain family 4, member 3), Cyp11b1 (cytochrome P450 11b1), Fn1 (fibronectin 1), Gal (Galanin), Hmox1 (hemeoxygenase 1), Hspb1 (heat shock protein b1), Igf1 (insulin like growth factor 2), Igf2 (insulin like growth factor 2), Il1b (interleukin 1 beta), Il16 (interleukin 16), Il18 (interleukin 18), IL1rn (interleukin 1 receptor antagonist), Mmp8 (matrix metallopeptidase 8), Mmp9 (matrix metallopeptidase 9), Niacr1 (niacin receptor 1), Ptgs2 (prostaglandin-endoperoxide synthase 2), S100a19 (S100 calcium binding protein A9), and S100a11 (S100 calcium binding protein A11). The RT-PCR data shown in the figure was normalized to the housekeeping gene β-actin.