Progesterone and vitamin D: Improvement after traumatic brain injury in middle-aged rats

Abstract

Progesterone (PROG) and vitamin D hormone (VDH) have both shown promise in treating traumatic brain injury (TBI). Both modulate apoptosis, inflammation, oxidative stress, and excitotoxicity. We investigated whether 21 days of VDH deficiency would alter cognitive behavior after TBI and whether combined PROG and VDH would improve behavioral and morphological outcomes more than either hormone alone in VDH-deficient middle-aged rats given bilateral contusions of the medial frontal cortex. PROG (16 mg/kg) and VDH (5 μg/kg) were injected intraperitoneally 1 h post-injury. Eight additional doses of PROG were injected subcutaneously over 7 days post-injury. VDH deficiency itself did not significantly reduce baseline behavioral functions or aggravate impaired cognitive outcomes. Combination therapy showed moderate improvement in preserving spatial and reference memory but was not significantly better than PROG monotherapy. However, combination therapy significantly reduced neuronal loss and the proliferation of reactive astrocytes, and showed better efficacy compared to VDH or PROG alone in preventing MAP-2 degradation. VDH+PROG combination therapy may attenuate some of the potential long-term, subtle, pathophysiological consequences of brain injury in older subjects.

Keywords: Aging; Combination treatments; Functional repair; Progesterone; Traumatic brain injury; Vitamin D deficiency; Vitamin D3 hormone.

Figure 1. Changes in body weight with both diets Changes in body weight with both diets

A. Weight curve with VitD-S diet. B. Weight curve with VitD-D diet. Body weights were measured on the day of surgery (day 0), and 1, 3, 7, 15, and 22 days post-surgery. Body weights decreased significantly after surgery except in Shams with both diets. There was no significant difference across the two diets.

Figure 2. Serum VDH level

A. Serum VDH level after 21 days’ diet conditioning. Rats fed a VitD-D diet had significantly lower serum VDH levels than those fed the VitD-S diet (p<0.01, n=5). B. Serum VDH levels of both diet cohorts at 22 days post-surgery. Serum VDH levels remained low at 22 days post-surgery within the VitD-D diet cohort compared to the VitD-S cohort (p<0.01, n=5). * compared with VitD-S, p<0.05.

Figure 3

Locomotor activity testing was done at 3 days (baseline) before surgery and at 3, 8, 15 and 21 days post-surgery or sham surgery. The average travel distance and resting time were presented as percentage of baseline. A. Travel distance of VitD-S cohort. B. Travel distance of VitD-D cohort. C. Resting time of VitD-S cohort. D. Resting time of VitD-D cohort.

Figure 4

Sticky task and grip strength tests were conducted at 3 days (baseline) before surgery and at 3, 8, 15 and 21 days post-surgery or sham surgery. The average latency to notice the sticker and grip strength were presented as percentage of baseline response. A. Latency to notice the sticker of VitD-S cohort. B. Latency to notice the sticker of VitD-D cohort. C. Grip strength of VitD-S cohort. D. Grip strength of VitD-D cohort.

Figure 5

Morris water maze testing began 10 days after TBI or sham surgery. Rats received 2 trials per day over 7 consecutive days of testing with a different starting locations. A–B. Time to find the platform in MWM training trial 1 each day with both diets. The latency to reach the platform in the Sham group was significantly better than that of the other treatment groups over time in trial 1 with both diets. The overall MWM latency of the Comb group was significantly better than that of the Veh group. C–D. Time to find the platform in MWM training trial 2. The results of trial 2 were consistent with trial 1 and showed that combination therapy improved acquisition of the task compared to Veh with both diets. E. A probe test without a platform was conducted on the 18^th day after surgery or sham surgery to evaluate time spent in the platform quadrant. With both diets, Veh rats spent significantly less time in the platform quadrant than Sham rats. PROG and Comb rats spent more time in the platform quadrant compared to Veh rats. There was no significant difference between PROG and Comb groups. There was no significant difference in probe test results across diet cohorts. *: compared to Sham with VitD-S, p<0.05; *: compared to Sham with VitD-D, p<0.05; ◊: compared to Veh with VitD-S, p<0.05; ♦: compared to Veh with VitD-D, p<0.05.

Figure 6

A. Infarct site. Brain sections from 6 coronal levels from bregma to 5 mm anterior to bregma were selected for Nissl staining 22 days after injury. Nissl staining showed that CCI resulted in substantial brain damage at the site of pre-frontal cortex. B. Necrotic cavity. All the CCI groups had more than 40 mm³ necrotic cavity which was significantly different from Sham groups with both diets (p<0.01). There were no significant differences in necrotic cavity among the CCI groups. C. Number of F-Jc positive cells. All the CCI groups with both diets had more delayed neuron death than the Sham groups. However, there were no significant differences among all CCI groups in delayed cell death. *, p<0.01, compared to Sham with VitD-S. #, p<0.01, compared to Sham with VitD-D.

Figure 7

A. Region of interest: Pictures were taken at the cortex close to the lesion and at the sub-cortex under the lesion to analyze the expression of MAP-2 and GFAP. B. GFAP expression in Sham. Immunofluorescence staining showed that at 22 days post-injury, the appearance of normal astrocytes in healthy cerebral cortex of Sham rats was evenly dispersed without overlapping (indicated by white arrows). C. Reactive astrogliosis in response to CCI. In the cortex of Veh animals at 22 days post-injury, immunofluorescence staining showed extensive overlaps and interdigitations of processes of severely reactive astrocytes on the border of the injury site (indicated by the arrow at upper left). There are other individual non-overlapping domains of reactive astrocytes with hypertrophy of the cell body and the processes near the injury site (indicated by the arrow at lower right).

Figure 8

GFAP/MAP-2 co-label staining in cortex close to the injury. A–J. GFAP-positive cells were labeled in red (Alexa Fluor 594 (ab’)) and MAP-2 in green (Alexa Fluor 488 (ab’)). The cell nuclei were labeled in blue by DAPI. K. The expression of GFAP in cortex close to the injury. *, p<0.01, compared to Sham withVitD-S. #, p<0.01, compared to Sham with VitD-D. L. MAP-2 expression in cortex of both diet cohorts. *, p<0.01, compared to Sham with VitD-S. #, p<0.01, compared to Sham with VitD-D.

Figure 9

GFAP/MAP-2 double staining in sub-cortex under the injury. A–J. GFAP-positive cells were labeled in red (Alexa Fluor 594 (ab’)) and MAP-2 in green (Alexa Fluor 488 (ab’)). The cell nuclei were labeled in blue by DAPI. K. The expression of GFAP in sub-cortex vertical to the injury of both diets. *, p<0.05, compared to Sham with VitD-S. #, p<0.05, compared to Sham with VitD-D. ◊, compared to Veh with VitD-S, p<0.05. ♦, compared to Veh with VitD-D, p<0.05. L: MAP-2 expression in sub-cortex of both diet cohorts. *, p<0.05, compared to Sham with VitD-S. #, p<0.05, compared to Sham with VitD-D. ◊, compared to Veh with VitD-S, p<0.05. ♦, compared to Veh with VitD-D, p<0.05; ▲: compared to PROG and VDH with VitD-S, p<0.05; ●: compared to PROG and VDH with VitD-D, p<0.05.