Vitamin D deficiency reduces the benefits of progesterone treatment after brain injury in aged rats

Abstract

Administration of the neurosteroid progesterone (PROG) has been shown to be beneficial in a number of brain injury models and in two recent clinical trials. Given widespread vitamin D deficiency and increasing traumatic brain injuries (TBIs) in the elderly, we investigated the interaction of vitamin D deficiency and PROG with cortical contusion injury in aged rats. Vitamin D deficient (VitD-deficient) animals showed elevated inflammatory proteins (TNFα, IL-1β, IL-6, NFκB p65) in the brain even without injury. VitD-deficient rats with TBI, whether given PROG or vehicle, showed increased inflammation and greater open-field behavioral deficits compared to VitD-normal animals. Although PROG was beneficial in injured VitD-normal animals, in VitD-deficient subjects neurosteroid treatment conferred no improvement over vehicle. A supplemental dose of 1,25-dihydroxyvitamin D(3) (VDH) given with the first PROG treatment dramatically improved results in VitD-deficient rats, but treatment with VDH alone did not. Our results suggest that VitD-deficiency can increase baseline brain inflammation, exacerbate the effects of TBI, and attenuate the benefits of PROG treatment; these effects may be reversed if the deficiency is corrected.

Fig. 1

Uninjured (SHAM) deficient animals show elevated levels of inflammatory cytokine proteins (light) compared to nutritionally normal animals (dark). Values are normalized to normal uninjured animals (vertical axis = 1). Asterisks denote a significant t-test with p < 0.05. Western blot images refer to the cytokine located in the graph above, and β-actin images indicate loading controls. Nrm: normal; Def: vitamin D deficient.

Fig. 2

(A) Injured deficient animals treated with vehicle show elevated levels of inflammatory proteins at 24 h (dark grey) and 72 h (light grey) after injury compared to nutritionally normal animals. (B) Injured deficient animals treated with PROG also show increased inflammation at 24 and 72 h compared to normal animals. All protein results are normalized to the values for nutritionally normal animals at the same time-point, i.e., all normal values (not shown) are at value = 1 on the vertical axis. Asterisks denote a significant t-test with p < 0.05. Western blot images refer to the cytokine located in the graph above, and β-actin images indicate loading controls. Nrm: normal; Def: vitamin D deficient.

Fig. 3

Levels of individual inflammatory cytokine proteins, cleaved caspase-3, and p53 in deficient injured animals under different treatment conditions at 24 h (dark grey) and 72 h (light grey) after injury. Results are normalized to the vehicle (VH) group within each time-point (vertical axis value = 1). Asterisks (*) denote post hoc p < 0.05 significance relative to VH, and (#) denotes p < 0.05 relative to PROG. The major treatment effect significantly different from vehicle in most cases is only D + PROG, suggesting a reversal of the injurious effect of deficiency. Western blot images refer to the treatment group in the graph above, and β-actin images indicate loading controls.

Fig. 4

Open-field activity results for normal (dark grey) and deficient (light grey) animals showing a beneficial effect with combined D + PROG treatment in all cases. All results are normalized to the SHAM group for each nutritional condition. Asterisks (*) denote post hoc p < 0.05 vs. VH (normal) and hash marks (#) denote p < 0.05 vs. VH (deficient).