Vitamin D Deficiency Exacerbates Experimental Stroke Injury and Dysregulates Ischemia-Induced Inflammation in Adult Rats

Robyn Balden; Amutha Selvamani; Farida Sohrabji

doi:10.1210/en.2011-1783

. 2012 Mar 9;153(5):2420–2435. doi: 10.1210/en.2011-1783

Vitamin D Deficiency Exacerbates Experimental Stroke Injury and Dysregulates Ischemia-Induced Inflammation in Adult Rats

Robyn Balden ¹, Amutha Selvamani ¹, Farida Sohrabji ^1,^✉

PMCID: PMC3339639 PMID: 22408173

Abstract

Vitamin D deficiency (VDD) is widespread and considered a risk factor for cardiovascular disease and stroke. Low vitamin D levels are predictive for stroke and more fatal strokes in humans, whereas vitamin D supplements are associated with decreased risk of all-cause mortality. Because VDD occurs with other comorbid conditions that are also independent risk factors for stroke, this study examined the specific effect of VDD on stroke severity in rats. Adult female rats were fed control or VDD diet for 8 wk and were subject to middle cerebral artery occlusion thereafter. The VDD diet reduced circulating vitamin D levels to one fifth (22%) of that observed in rats fed control chow. Cortical and striatal infarct volumes in animals fed VDD diet were significantly larger, and sensorimotor behavioral testing indicated that VDD animals had more severe poststroke behavioral impairment than controls. VDD animals were also found to have significantly lower levels of the neuroprotective hormone IGF-I in plasma and the ischemic hemisphere. Cytokine analysis indicated that VDD significantly reduced IL-1α, IL-1β, IL-2, IL-4, IFN-γ, and IL-10 expression in ischemic brain tissue. However, ischemia-induced IL-6 up-regulation was significantly higher in VDD animals. In a separate experiment, the therapeutic potential of acute vitamin D treatments was evaluated, where animals received vitamin D injections 4 h after stroke and every 24 h thereafter. Acute vitamin D treatment did not improve infarct volume or behavioral performance. Our data indicate that VDD exacerbates stroke severity, involving both a dysregulation of the inflammatory response as well as suppression of known neuroprotectants such as IGF-I.

Vitamin D is one of the oldest hormones, involved not only in calcium and bone homeostasis but also known to regulate genes involved with immunomodulation, proliferation, and regulation of cell growth and differentiation (1–4). Although the canonical synthetic pathway of activated vitamin D hormone, also known as calcitriol or 1,25-OH₂-vitamin D₃, ends in the kidney, many other cell types, such as vascular smooth muscle cells, microglia (5, 6), astrocytes (7), and cerebral neurons (7), synthesize 1-α-hydroxylase (CYP27B1), the final enzyme in the activation pathway, to elevate local levels of 1,25-OH₂D₃ (4, 5, 8, 9). Furthermore, vitamin D receptors (VDR) are widely expressed, including endothelium, activated monocytes and T cells (2, 10–12), astrocytes, cardiomyocytes (13), and neurons.

Although vitamin D research has increased remarkably in the last decade (2, 14–20), the putative benefits of this hormone remain to be definitively determined. Low vitamin D levels have been implicated in numerous chronic inflammatory conditions, including cardiovascular disease (CVD), cancer, autoimmune, and infectious diseases (2, 5, 16, 21–23). In the elderly population, vitamin D deficiency (VDD) is common and likely contributes to the increased pathogenesis and incidence of CVD and exacerbates these diseases once instated (24). Critical risk factors for cerebral infarction, such as age and hypertension, are associated with low serum 25-hydroxyvitamin D (25-OHD), the circulating form of vitamin D (25–27), and low serum 25-OHD is independently predictive of future strokes (24, 28). However, recent metaanalysis of randomized-controlled clinical trials investigating vitamin D supplements did not find a statistically significant benefit in reducing CVD risk or mortality (18, 29). Although multiple studies have found that vitamin D supplements may reduce CVD and mortality risk (30–32), evidence suggests that vitamin D supplementation is more important for at-risk populations than in generally healthy groups (33). For example, hypertensive subjects administered vitamin D in clinical trials experienced a blood pressure-lowering effect (27, 34–36). However, studies in which normotensive subjects were administered vitamin D saw no effects on blood pressure (18, 37).

Stroke is a leading cause of morbidity and mortality (38) and, with CVD, is the leading cause of death of both men and women (39) in the United States. Low vitamin D levels are associated with a greater risk for stroke, and experimental studies indicate that vitamin D can protect neurons from excitotoxic injury, similar to that seen in stroke. Cerebral ischemia has been shown to activate the VDR, resulting in increased nuclear translocation in an animal stroke model as well as elevation of VDR in human stroke patients (40). In vitro, vitamin D₃ protects rat cortical neurons (41) and retinal ganglion cells (42) against glutamate-induced neurotoxicity and cortical neurons against cyanide-induced neurotoxicity (43). Pretreatment, but not concurrent treatment, with 1,25-OH₂D₃ also reduced cell death in rat mesencephalic neuronal cultures exposed to reactive oxygen species (44). Furthermore, vitamin D enhances endogenous antioxidant pathways, such as γ-glutamyl transpeptidase (45), and reduces inducible nitric oxide synthase (46), suggesting a mechanism for vitamin D-induced neuroprotection. Additionally, vitamin D up-regulates neurotrophic factors, such as nerve growth factor, neurotrophin-3, neurotrophin-4 (47, 48), IGF-I (49), and glial cell line-derived neurotrophic growth factor (50). Growth factors, such as IGF-I, contribute to central nervous system (CNS) repair by promoting axon and dendrite sprouting and regrowth after stroke, which is a critical component of recovery of function (51), and also support the growth and survival of neural progenitor cells and other support cells, such as astrocytes and microglia, that play an integral role poststroke recovery (52). Similarly, vitamin D₃ has been shown to suppress inflammatory cytokines, such as TNF-α, IL-6, and nitric oxide in a microglial cell line (53), inhibit cluster of differentiation 4, major histocompatibility complex class III, and nitric oxide synthase in an experimental model of multiple sclerosis (46), and reduce ischemia-induced brain damage (54). Thus, vitamin D may promote cell survival through protrophic and antiinflammatory gene regulation.

Little is known about the role of VDD in the context of brain function and injury. Long-term vitamin D deprivation changes vasomotor reactivity in rats (55) and has profound effects on the anatomy of the developing brain (56), as well as the expression of critical growth factors and genes regulating oxidative phosphorylation, calcium homeostasis, synaptic plasticity, and neurotransmission (56, 57). In adult animals, mice maintained on a VDD diet were found to have impaired inflammatory cytokine production in activated macrophages (58). The effect of VDD on stroke severity, however, has not been studied. In the present study, we maintained animals on a VDD diet, and our data indicate that VDD increases stroke severity and sensory motor impairment, attenuates growth factor expression, and alters the profile of inflammatory cytokines.

Materials and Methods

Animals

Adult Sprague Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN), as mature adult females (4–5 months, 250 g) or middle-aged adult females (9–11 months, 280–350 g). Animals were maintained in a constant 12-h light, 12-h dark cycle, and food and water were available ad libitum. All procedures were in accordance with the National Institutes of Health and institutional guidelines governing animal welfare. For surgeries and tissue collection, the rats were deeply anesthetized (ketamine, 87 mg/kg; xylazine, 13 mg/kg).

Vitamin D treatment

Animals were assigned to VDD or vitamin D treatment groups, respectively, or a control group.

VDD studies

Animals were fed ad libitum control rat chow (diet no. 89124; Harlan Teklad, Madison, WI) or VDD chow (diet no. 89123, Harlan Teklad) for 8 wk. Control and VDD chow were otherwise equivalent in composition, including calcium and phosphorous levels. Individual body weight and diet consumption per cage were recorded weekly, and no differences were observed between groups.

Vitamin D treatment studies

To effectively solubilize and deliver vitamin D₃ in vivo, 2-hydroxypropyl-β-cyclodextrin (Sigma, St. Louis, MO), an organic cyclic caging molecule, was used as a vehicle as described in Cekic et al. (59). Animals were injected with ip with 22.5% 2-hydroxypropyl-β-cyclodextrin solution alone (controls) or 22.5% 2-hydroxypropyl-β-cyclodextrin + 10 μg/kg vitamin D₃ (Sigma), 4 h after middle cerebral artery occlusion (MCAo) and every 24 h thereafter for 5 d.

Middle cerebral artery occlusion

Animals were subjected to stereotaxic surgery to occlude the left MCA according to a previously established laboratory protocol (60). Briefly, anesthetized animals were placed in a stereotaxic frame, and a small hole was drilled in the skull at the following coordinates relative to bregma: +0.9 mm anterior posterior, +3.0 mm medial lateral. A needle, attached to syringe containing endothelin-1 (ET-1), was lowered to a depth of −8.0 mm relative to the dura. ET-1 was delivered slowly to the MCA at the rate of.02 μl/30 sec, for a total of 3 μl (0.5 μg/μl in PBS; American Peptide Co., Sunnyvale, CA). This experimental stroke model results in a prolonged ischemia and gradual reperfusion, which lasts approximately 7 h in the striatum and 16 h in the cortex (61). Optical imaging studies using a small fluorescent probe (Cy5.5; 1 kDa molecular mass) indicated that ET-1-induced vasoconstriction is clearly visible 5 h after ET-1, whereas reperfusion is apparent at 10 h in the midline (striatum) and is fully reestablished when imaged at 21 h after ET-1 (62). During surgery, the rats were maintained at 37 C throughout, and oxygen saturation and respiratory rate were constantly monitored using the Mouse Oximeter (STARR Life Sciences, Oakmont, PA). There were no differences in oxygen saturation (controls, 85.99 ± 1.477; VDD, 85.05 ± 1.95) and respiratory rate (control, 55.76 ± 3.78; VDD, 56.69 ± 4.78) between the treatment groups. At termination, the brain was rapidly removed, hand sectioned using a brain mold, and slices were processed for 2,3,5-triphenyl-tetrazolium chloride (TTC) (Sigma) staining and biochemical analysis.

Sensorimotor tests

The vibrissae-elicited forelimb placing test was performed before and after MCAo surgery to assess the functional effects of neurovascular injury, according to our published protocol (60, 62). Animals were subject to same-side placing trials and cross-midline placing trials elicited by stimulating the ipsi- and contralesional vibrissae. Ten trials each were performed for the ipsi- and contralesional vibrissae, and another experimenter recorded scoring during trials. Trials in which the animal seemed to struggle or make premature forelimb movements were not counted.

Tape test

The tape test is also a sensitive indicator of sensorimotor deficit in ischemic injury (63). Two pieces of adhesive-backed foam tape (1 × 1/2″) were used as bilateral tactile stimuli attached to the palmar surface of the paw of each forelimb. For each forelimb, the investigator recorded the time it took to remove each stimulus (tape) from the forelimbs during three trials per day for each forepaw. Animals were allowed to rest for 1 min between sessions, and each test session had a maximum time limit of 120 sec.

Infarct volume

Brain slices (2 mm thick) between −2.00 and +4.00 mm anterior posterior from bregma were incubated in a 2% TTC solution at 37 C for 30 min and later photographed using a digital camera attached to a dissecting microscope (Olympus, New York, NY). Infarct volume was determined from digitized images using the Quantity One software package (Bio-Rad, Hercules, CA). Typically, four such slices were used for analysis. The area of the cortical and striatal infarct was measured separately in all slices as well as the contralateral hemisphere. Details of volume determination are described in Ref. 60. The volume of the infarct was normalized to the volume of the contralateral (nonoccluded) hemisphere. To ensure reliable and consistent detection of the infarct zone, images were digitally converted to black-and-white and magnified, and all traces were performed by the same investigator (R.B.).

Tissue collection

For biochemical analyses, cortical and striatal tissue from the left and right hemispheres and splenic tissue was collected and stored at −80 C. Tissue was later processed for protein extraction, and protein concentrations were determined using the bicinchoninic acid method (Pierce, Rockford, IL) with BSA as a standard (64). Blood was centrifuged at 2500 rpm for 30 min to collect the plasma.

Vitamin D levels

Plasma concentrations of circulating 1,25-OH₂ vitamin D, 25-OH vitamin D, and local 25-OHD concentrations in homogenized tissues were determined by ELISA (Immunodiagnostic Systems, Fountain Hills, AZ), according to manufacturer's instructions. For 1,25-OH₂ vitamin D, briefly, controls and samples were delipidated and transferred to supplied immunocapsules containing monoclonal antibody to 1,25-dihydroxyvitamin D in suspension with vitamin D-binding protein inhibitor for immunoextraction. After eluting calcitriol from the immunocapsule gel, samples were evaporated in borosilicate glass tubes in a heating block for 30 min under nitrogen gas flow. Evaporated samples were reconstituted in assay buffer and incubated overnight with 100 μl of primary antibody solution. Standards, controls, and samples were then pipetted into a 96-well plate precoated with antibodies specific for 1,25-OH₂D and incubated at room temperature for 90 min on an orbital plate shaker. After washes, the remainder of the assay was completed according to the same ELISA protocol as for 25-OHD. For 25-OHD, briefly, standards, controls, and samples were pipetted into a 96-well plate precoated with antibodies specific for 25-OH vitamin D and incubated at room temperature for 2 h. After washes, plates were incubated with 200 μl of streptavidin peroxidase for 30 min. After the next set of washes, 200 μl of stabilized chromogen were added to each well and incubated for an additional 30 min. The color reaction was stopped by an equal volume of stop solution and read at 450 nm (650-nm reference wavelength) in a microplate reader (Bio-Tek, Winooski, VT). Standard curves were established from optical densities of wells containing known dilutions of standard using KC3 software (Bio-Tek), and sample measurements were interpolated from standard curves. For homogenized tissues, the results of these assays were normalized for individual sample protein content. The lower detection limit of the ELISA is 6 nmol/liter as indicated by the manufacturer.

IGF-I levels

Concentrations of circulating IGF-I in plasma and local IGF-I in homogenized tissues was determined by ELISA (R&D Systems, Minneapolis, MN), according to manufacturer's instructions. Briefly, standards, controls, and samples were pipetted into a 96-well plate precoated with antibodies specific for IGF-I and incubated at room temperature for 2 h. After washes, plates were sequentially incubated with 100 μl of conjugate for 2 h. After the next set of washes, 100 μl of substrate solution were added to each well and incubated for an additional 30 min. The color reaction was stopped by an equal volume of stop solution and read at 450 nm (540-nm reference wavelength) in a microplate reader (Bio-Tek). Standard curves were established from optical densities of wells containing known dilutions of standard using KC3 software (Bio-Tek), and sample measurements were interpolated from standard curves. For homogenized tissues, the results of these assays were normalized for individual sample protein content.

Quantitative RT-PCR

Quantitative RT-PCR was performed for CYP27B1, 24-hydroxylase (CYP24A1), and IGF-I in brain tissue, and for IGF-I in liver tissue according to established laboratory protocol. Briefly, total RNA was extracted from homogenized tissues using an RNeasy kit (QIAGEN, Valencia, CA) according to manufacturer's directions. Total RNA for each sample was quantitated using NanoDrop technology (Thermo Scientific, Waltham, MA), and normalized total RNA samples were added to a solution containing qScript cDNA SuperMix (Quanta BioSciences, Gaithersburg, MD). SYBR Green PCR Master Mix (Molecular Probes, Inc., Eugene, OR)-based RT-PCR using MyiQ single-color RT-PCR detection system (Bio-Rad) was used to quantify the following: CYP27B1, CYP24A1, and IGF-I (Real Time Primers, Elkins Park, PA), and cyclophilin (Invitrogen, Carlsbad, CA). Forward and reverse primer sequences, respectively, for CYP24A1, 5′-TGGGTGAATACGCTCTACCC-3′ and 5′-TATCCAGCAGAGAGCCAGGT-3′; for CYP27B1, 5′-AAGGCAGCTGTCATCATCTC-3′ and 5′-TCTGAAGGCTTTGGATCTTG-3′; and for IGF-I, 5′-TCTGCTTGCTCACCTTTACC-3′ and 5′-TACATCTCCAGCCTCCTCAG-3′.

Cytokine assay

Cytokine expression in the brain and spleen was measured after MCAo using a cytokine multiplex ELISA (Milliplex rat MAP assay system; Millipore, Bedford, MA) in conjunction with the Bio-Plex Suspension Array Systems (Bio-Rad) with High-Throughput Fluidics, following procedures described in Lewis et al. (65). The results of these assays were normalized for individual sample protein content using data collected from the bicinchoninic acid procedure (described previously). Briefly, standards, controls, and samples were pipetted into a 96-well plate, followed by the addition of 25 μl of antibody-immobilized beads specific for rat granulocyte-macrophage colony stimulating factor, IL-1α, monocyte chemotactic protein-1, IL-4, IL-1β, IL-2, IL-6, IL-10, IL-12p70, IL-5, IFN-γ, IL-18, growth-related oncogene-KC, and TNF-α. After washes, plates were sequentially incubated with 25 μl of detection antibody, followed by the addition of 25 μl of streptavidin-phycoerythrin. Standard curves were established from median fluorescent intensities of wells containing known dilutions of standard, and sample measurements were interpolated from standard curves.

TGF-β1 levels

Concentrations of local TGF-β1 (which is not available as part of the multiplex assay) was determined by singleplex ELISA (Milliplex rat MAP assay system; Millipore), according to manufacturer's instructions and in conjunction with the Bio-Plex Suspension Array Systems (Bio-Rad) with High-Throughput Fluidics, following procedures described in Lewis et al. (65).

Statistical analysis

Statistical analysis was performed using a statistical software package (SPSS, Inc., Chicago, IL), and group differences were considered significant at P ≤ 0.05. For most measures, data were analyzed using a two-way ANOVA coded for diet or treatment as an independent variable (VDD and normal rat chow or 10 μg/kg vitamin D₃ + vehicle and vehicle alone) and brain region as a repeated measure (cortex and striatum). The data are expressed as the mean ± sem for VDD and control mature adults or vitamin D treatment and control middle-aged adult females for each study. All experimental groups had an n = 4–7, which is indicated in each figure legend.

Results

VDD diet studies

Weight analysis

Average weight and diet consumption were similar in animals consuming VDD chow as compared with animals fed control chow over the 8-wk period. Starting weights were 262.06 ± 15.9 g for the control group and 264.8 ± 23.0 g in the VDD group. After the diet treatment period, final weights were 287.5 ± 19.1 and 278.45 ± 21.41 g, respectively, for control and VDD groups.

Local and circulating levels of 1,25-OH₂ vitamin D and 25-OH vitamin D

As shown in Fig. 1, A and B, VDD diet significantly reduced circulating vitamin D (25-OHD) levels in plasma to less than 22% of control levels (P < 0.05) and circulating activated vitamin D (1,25-OH₂D) levels to less than 33% of control levels (P < 0.05), respectively. In the brain, 25-OHD levels were significantly and transiently suppressed in the cortex [F_(1,11), 6.73; P < 0.05] and striatum [F_(1,12), 11.84; P < 0.05] of VDD groups compared with chow controls, at 3 d after stroke (Fig. 1C). Furthermore, vitamin D levels were higher in the ischemic cortex [F_(1,11), 38.15; P < 0.05] and striatum [F_(1,11), 24.36; P < 0.05] compared with the contralateral side. At 5 d after stroke, vitamin D levels remained elevated in the ischemic cortex [F_(1,7), 11.19; P < 0.05] but not in the striatum (Fig. 1D). For comparison, 25-OHD levels were measured in the small intestine, an unrelated tissue classically associated with vitamin D function. No group difference in intestinal tissue 25-OHD levels was observed, and local 25-OHD levels were similar in brain and intestinal tissue (Fig 1, D and E).

Local expression of vitamin D-associated genes

Although circulating vitamin D was reduced by VDD diet, local vitamin D levels in brain tissue were not. To clarify the discrepancy between large group differences in circulating 25-OHD and 1,25-OH₂D levels and virtually no diet-related differences in brain 25-OHD levels (Fig. 1D), expression of the activating CYP27B1 and inactivating CYP24A1 genes was analyzed in brain tissue. As shown in Fig. 1F, VDD diet significantly increased CYP24A1 expression in the ischemic cortex and striatum [F_(1,10), 6.98; P < 0.05] compared with control diet at 5 d after stroke. Furthermore, ischemia led to a much greater elevation of CYP24A1 levels in the injured hemisphere as indicated by a significant effect of ischemia [F_(1,10), 25.11; P < 0.05]. For CYP27B1, no significant differences were found (Fig. 1G).

Infarct volume

Animals were subject to ET-1-induced MCAo after 8 wk of specialized diets and were euthanized 3 or 5 d after stroke. A cortical-striatal infarct was observed in animals of both control and VDD groups, shown in TTC-stained sections in Fig. 2A (3 d after stroke) and Fig. 2B (5 d after stroke). At 3 d after stroke, infarct volume was not significantly different between the control diet and VDD groups (Fig. 2C). However, at 5 d after stroke, the volume of stroke tissue had partially improved in controls, whereas VDD animals had little or absent improvement. Thus, there was a significant increase in infarct volume [F_(1,7), 5.85; P < 0.05] due to VDD, with greater cortical (28%) and striatal (126%) damage in the VDD group (Fig. 2D).

Fig. 2. — Effect of VDD diet on infarct volume. Animals maintained on a VDD diet or control diet were subject to MCAo and euthanized 3 or 5 d later. Representative slices of TTC-stained sections depict the rostrocaudal extent of the infarct in control and VDD animals 3 d (A) and 5 d (B) after ischemia. Unstained tissue is indicative of dead or infarcted tissue. Histograms depict mean ± sem of infarct volume in the cortex and striatum normalized to the contralateral side. C, At d 3, there were no significant differences in cortical or striatal infarct volume between the control diet and VDD animals. D, By 5 d after stroke, both cortical and striatal infarct volumes were significantly larger in the VDD group compared with the control diet-fed animals (a, Main effect of diet). Striatal volumes were more severely affected by VDD (c, Interaction effect of diet treatment and ischemia). Data were analyzed by two-way ANOVA for diet treatment and ischemia for each region; a, Main effect of diet; b, main effect of ischemia; c, interaction effect of diet treatment and ischemia. Group differences were considered significant at P ≤ 0.05. *Bars* represent mean ± sem; n = 4–7 in each group. Vctx, Volume cortex; Vstr, volume striatum; Vc, volume contralateral hemisphere.

Behavioral outcomes

The vibrissae-elicited forelimb placement task evaluates functional capabilities dependent on the cortex and striatum and interhemispheric integration between these two regions. Forelimb placement was completely accurate before stroke surgery. At 3 d after stroke, significant functional deficits were evident in both diet groups. However, these deficits were more severe in the VDD animals vs. controls. In the cross-midline vibrissae-elicited forelimb placement test (Fig. 3A), VDD animals performed significantly worse than controls for the contralesional paw [F_(1,11), 6.05; P < 0.05]. This finding was confirmed and extended by the tape test, a sensitive indicator of sensorimotor deficit in ischemic injury (52). As expected, no significant pre- and poststroke differences were seen in the amount of time needed to remove the tape from the ipsilesional paw. However, significant delays were seen in the amount of time needed to remove the tape from the contralesional paw after stroke [F_(1,12), 84.79; P < 0.05] (Fig. 3B). Furthermore, VDD animals took longer to remove the tactile stimulus from the contralesional paw compared with controls before ischemic injury, and this was further amplified after stroke, where animals in the VDD group simply failed to remove the tape during the testing duration [F_(1,12), 6.88; P < 0.05].

Fig. 3. — Effect of VDD diet on behavioral performance. All animals were assessed before and 3 d after stroke on two behavioral tasks. A, Histograms depict mean (±sem) percent correct responses on the cross-midline vibrissae-elicited forelimb placement task, pre- and postischemia. Performance on the left (ipsilesional) limb and right (contralesional) limb was significantly affected by ischemia (b, Main effect of ischemia). However, on the right limb, VDD diet treatment (*green bar*) significantly worsened performance after stroke compared with the control diet (*blue bar*) (c, Interaction effect of ischemia and diet). B, Tape test. Histograms depict mean (±sem) duration (in seconds) to remove tape from the palmar surface of the front limb. Performance on this task was significantly impaired after stroke compared with prestroke in both diet groups (b, Main effect of ischemia). Moreover, mean duration to remove the tape was also affected by diet, such that VDD diet groups took longer to remove the tape, or did not remove it at all during the allotted time, compared with the control diet animals (a, Main effect of diet). Data were analyzed by two-way ANOVA for pre-/postischemia and diet treatment for each limb; a, Main effect of diet; b, main effect of ischemia; c, interaction effect of diet treatment and ischemia. Group differences were considered significant at P ≤ 0.05. *Bars* represent mean ± sem; n = 6–7 in each group.

Local and circulating levels of IGF-I

IGF-I expression was determined by ELISA in plasma and brain tissue. Plasma IGF-I is significantly higher in uninjured (prestroke) control animals (1435.1 ± 27.15 pg/ml) compared with VDD rats (746.18 ± 116.03 pg/ml). IGF-I levels were virtually identical in control and VDD groups 3 d after ischemia. However, by 5 d after ischemia, plasma IGF-I levels were 38.5% lower in the VDD group compared with controls (Fig. 4A). Brain IGF-I levels were elevated, as expected, in both the ischemic cortex and striatum as compared with nonischemic tissues at 3 d [F_(1,12), 119.97; P < 0.05] and 5 d after ischemia [F_(1,6), 67.24; P < 0.05]. No diet-induced differences were seen in IGF-I levels at 3 d after stroke (Fig. 4B). However, at 5 d after ischemia, IGF-I levels were further elevated in the control group, whereas VDD groups maintained a similar level of IGF-I seen at 3 d [F_(1,6), 16.31; P < 0.05] (Fig. 4C), indicating that VDD attenuates elevated levels of this neuroprotectant. To determine whether the changes in serum and brain tissue IGF-I levels were due to a local or systemic effect, IGF-I levels were measured in the liver, the largest source of IGF-I. In keeping with circulating and brain tissue IGF-I levels, VDD liver tissue had significantly lower IGF-I expression as compared with control liver (P < 0.05) (Fig. 4E). However, IGF-I levels in the spleen were no different in VDD vs. controls, indicating differential tissue regulation of IGF-I by VDD (Fig. 4G). Additionally, IGF-I mRNA levels were also measured in the brain. As shown in Fig. 4D, IGF-I mRNA levels were significantly elevated on the ischemic side (both cortex and striatum). However, there was no difference in IGF-I gene expression between control and VDD groups. Collectively, these data indicate that poststroke brain IGF-I levels reflect a combination of local and systemic IGF-I levels and that diet-induced differences in IGF-I in the brain is likely due to influx of circulating peptide.

Fig. 4. — Effect of VDD diet on circulating and local IGF-I. A, Plasma IGF-I levels were measured at 3 and 5 d after ischemia in control and VDD female rats. At 3 d after stroke, plasma IGF-I levels were similar in VDD and control groups. However, by 5 d after stroke, IGF-I levels were sustained in control diet animals, whereas peptide levels were decreased in the VDD diet group. Thus, circulating IGF-I levels were reduced 38.5% of the levels observed in control chow-fed animals (*, P < 0.05). Data points represent mean ± sem. B, IGF-I levels in the brain 3 d after ischemia in VDD and control animals. Lysates from the ischemic (*left*) hemisphere had significantly greater IGF-I expression compared with the nonischemic (*right*) hemisphere (b, Main effect of ischemia). Diet did not affect IGF-I levels in either the cortex or striatum at 3 d after ischemia. C, At 5 d after ischemia, local IGF-I levels were still elevated in the ischemic hemisphere compared with the ischemic side (b). However, at 5 d after stroke, IGF-I levels were significantly elevated in the cortex and striatum of control animals compared with VDD animals (c, Interaction effect of diet treatment and ischemia). VDD attenuated IGF-I levels in ischemic cortical (75.5%) and striatal (47.2%) tissues. D, IGF-I mRNA expression in the brain 5 d after stroke. Similar to IGF-I protein, IGF-I mRNA levels were significantly greater in the ischemic hemisphere (b). However, diet did not affect IGF-I mRNA levels in the cortex or striatum. E and F, Liver expression of IGF-I. At 5 d after ischemia, IGF-I protein (E) and mRNA (F) levels were significantly reduced (35.14 and 28.0%, respectively) in VDD liver tissue as compared with liver from controls (*, P < 0.05). Data points represent mean ± sem. For comparison, IGF-I levels in the spleen, a peripheral lymphoid organ known to respond to hypoxia, are shown 5 d after stroke. G, Unlike liver tissue, splenic IGF-I levels were not affected by diet. Data were analyzed by three-way ANOVA for diet treatment, ischemia, and region as repeated measures; a, Main effect of diet; b, main effect of ischemia; c, interaction effect of diet treatment and ischemia. Group differences were considered significant at P ≤ 0.05. *Bars* represent mean ± sem; n = 4–7 in each group.

Effects of VDD on the central ischemic inflammatory response

The impact of the VDD diet on cerebral inflammation was assessed by measuring a panel of cytokines/chemokines in cortical and striatal brain tissue from VDD and control animals. In both groups, MCAo increased overall cytokine expression in the cortex and striatum of the ischemic hemisphere as compared with that of the nonischemic hemisphere (data not shown). However, baseline (nonischemic) and ischemia-induced cytokine expression was generally decreased in VDD animals as compared with controls. The table in Fig. 5A summarizes the differences in cytokine levels at 5 d after ischemia in VDD animals relative to controls. Specifically, VDD animals expressed significantly less IL-1α, IL-1β, IL-2, IL-4, and IFN-γ in response to cortical ischemia as compared with control animals, whereas reducing IL-1α and IL-10 in the striatum (P < 0.05). IL-6 was the only cytokine that was elevated in ischemic tissue in the VDD group compared with controls. As shown in Fig. 5B, ischemia elevated IL-6 levels in the cortex [F_(1,6), 80.31; P < 0.05] and striatum [F_(1,6), 5.97; P < 0.05] compared with the nonischemic side. Furthermore, in both regions, VDD led to a greater elevation of ischemia-induced IL-6 as indicated by a significant interaction effect of ischemia and diet in cortex [F_(1,6), 9.08; P < 0.05] and striatum [F_(1,6), 10.38; P < 0.05]. Ischemia also elevated TGF-β1 levels in the cortex [F_(1,6), 58.35; P < 0.05] and striatum [F_(1,6), 13.57; P < 0.05] compared with the nonischemic side (Fig. 5C). TGF-β1 levels were significantly correlated with IL-6 levels in the ischemic cortex (r = 0.81; P < 0.05) and striatum (r = 0.78; P < 0.05).

Fig. 5. — Postischemia brain and spleen cytokine expression in control and VDD rats. Brain samples from the ischemic (*left*) hemisphere and nonischemic (*right*) hemisphere were collected at d 5 after MCAo and analyzed for cytokine/chemokine expression. A, *Arrows* indicate the direction of significant changes in expression in specific cytokines in VDD animals relative to controls (P ≤ 0.05). B, At 5 d after stroke, IL-6 levels were significantly higher in the ischemic cortex and striatum compared with the nonischemic side (b, Main effect of ischemia). Furthermore, VDD resulted in a greater elevation of IL-6 in both the cortex (1.21-fold) and striatum (1.51-fold) compared with controls (c, Interaction effect of diet treatment and ischemia). C, At 5 d after stroke, TGF-β1 levels were significantly higher in the ischemic cortex and striatum compared with the nonischemic side (b, Main effect of ischemia). Data were analyzed by two-way ANOVA for diet treatment and ischemia; for each region; b, Main effect of ischemia; c, interaction effect of diet treatment of ischemia. Group differences were considered significant at P ≤ 0.05. *Bars* represent mean ± sem; n = 4–7 in each group. GM-CSF, Granulocyte-macrophage colony stimulating factor.

Effects of VDD on the peripheral ischemic inflammatory response

The spleen is known to mobilize its reservoirs of immune cells as part of the early response to inflammatory and hypoxic stimuli, such as cerebral ischemia (66–68), resulting in decreased splenic weight in the acute phase of stroke. This finding was confirmed in our model, where normalized splenic weights were inversely correlated with cortical infarct volume (r = −0.67, P < 0.05). Cytokine expression in the spleen was also assessed in VDD and control animals. Although cytokine levels in VDD ischemic brain tissue were generally decreased, specific cytokines in VDD splenic tissue were up-regulated as compared with levels in the control group. As Fig. 5A illustrates, IL-1α, IL-4, and IL-5 cytokine levels in splenic tissue from VDD animals were significantly higher than control levels by d 5 (P < 0.05). These data indicate that the spleen responds to ischemia by mounting peripheral immune mobilization.

Acute vitamin D treatment studies

To determine the effect of acute vitamin D administration on ischemic injury, middle-aged female animals subject to ET-1-induced stroke were injected ip with 10 μg/kg vitamin D₃ + vehicle or vehicle alone after stroke. As shown in Fig. 6A, vitamin D₃ injections consistently elevated (3.9-fold) plasma 25-OHD levels significantly above those of animals injected with vehicle alone (P < 0.05). However, local 25-OHD levels in brain tissue (Fig. 6B) from vitamin D₃-treated and control groups were not significantly regulated by vitamin D injections in the cortex [F_(1,7), 0.45; P > 0.05] or striatum [F_(1,7), 0.30; P > 0.05]. Furthermore, ischemia-induced up-regulation of local 25-OHD levels was seen in both the cortex [F_(1,7), 7.65; P < 0.05] and striatum [F_(1,7), 33.23; P < 0.05] as compared with contralateral nonischemic tissues, similar to that seen in the VDD study (Fig. 4B).

Fig. 6. — Levels of plasma and brain 25-OH vitamin D levels after vitamin D₃ injections. Plasma and brain tissue was derived from control and vitamin D-treated female rats after five consecutive days of vehicle or vitamin D₃ + vehicle ip injections, respectively. Samples were collected at 5 d after ischemia. A, Vitamin D treatments significantly increased circulating vitamin D, 25-OHD, levels (3.9-fold) as compared with levels in control rats injected with vehicle alone (*, P < 0.05). *Bars* represent mean ± sem. B, Brain 25-OHD levels, however, were not significantly affected by acute ip vitamin D₃ treatments. There was a small but significant elevation of 25-OHD in ischemic tissues compared with nonischemic tissues. Additionally, in the striatum, 25-OHD levels were affected by an interaction of vitamin D treatment and ischemia, such that 25-OHD levels in the ischemic striatum were significantly higher in the control group compared with the vitamin D-treated animals. Data were analyzed by two-way ANOVA for hormone treatment and ischemia for each brain region; b, Main effect of ischemia; c, interaction effect of ischemia and hormone treatment. *Bars* represent mean ± sem; n = 4–5 in each group.

Effect of acute vitamin D₃ treatment on infarct volume.

To determine the neuroprotective effects of vitamin D₃ treatment, animals were subject to MCAo surgery followed by acute poststroke treatments of 10 μg/kg vitamin D₃ + vehicle or vehicle alone. Hormone (or vehicle) treatment was initiated 4 h after stroke and then given every 24 h thereafter for 5 d. Animals were then terminated and assessed for infarct volume. Representative TTC-stained brain slices from control and vitamin D-treated animals are shown in Fig. 7A, and quantitative analysis of infarct volume, depicted in the histogram in Fig. 7B, indicated no significant differences between vehicle and vitamin D-injected groups.

Fig. 7. — Effect of vitamin D₃ injections on infarct volume. Animals were subject to MCAo and euthanized 5 d later. A, Representative slices of TTC-stained sections depict the rostrocaudal extent of the infarct in control and vitamin D₃-treated animals. B, Histograms depict infarct volume normalized to the contralateral side. Infarct volume was not affected by acute vitamin D₃ treatment in the cortex or the striatum. *Bars* represent mean ± sem. C, Performance on sensory motor task before and after ischemia. Animals were tested before and after ischemia on the vibrissae-evoked forelimb placement task. Histogram depicts mean (±sem) correct responses on the cross-midline vibrissae-elicited forelimb placement task for each forelimb. Postischemic performance was significantly affected on the left and right limb. However, vitamin D₃ treatment had no affect on the performance of either limb. Data were analyzed by two-way ANOVA for pre-/postischemia and vitamin D₃ treatment; b, Main effect of ischemia. Group differences were considered significant at P ≤ 0.05; n = 4–5 in each group.Vctx, Volume cortex; Vstr, volume striatum; Vc, volume contralateral hemisphere.

Behavioral analysis

Consistent with the lack of vitamin D₃ treatment effect on infarct volume, hormone treatment did not differentially affect forelimb placement as compared with control animals. The same-side paw-placing reflex task for the contralesional limb, in which both groups of animals performed 100% before MCAo, showed a significant loss (100%) of paw-placing behavior when the vibrissae contralateral to the lesion side was stimulated after MCAo (P < 0.05). The cross-midline task, which requires increased interhemispheric integration as compared with the same-side task, was significantly affected by MCAo in both forelimbs of vitamin D-treated and control animals [F_(1,7), 91.53 for the left limb; F_(1,7), 293.37 for the right limb; P < 0.05] (Fig. 7C). Vitamin D₃ injections did not improve sensory motor function.

Local and circulating levels of IGF-I

Acute poststroke vitamin D treatments differentially affected IGF-I levels in plasma and brain tissue. Analysis of plasma revealed significantly higher IGF-I levels (39.5%) in vitamin D-treated middle-aged animals as compared with controls (P < 0.05) (Fig. 8A). Thus, vitamin D availability is strongly associated with plasma IGF-I levels as indicated both by reduced circulating levels of IGF-I in VDD female animals and elevated IGF-I plasma levels in vitamin D-treated females as compared with control animals. Conversely, vitamin D treatments prevented ischemia-induced up-regulation of local IGF-I expression in ischemic brain tissue, as illustrated in Fig. 8B. Although IGF-I levels were elevated as expected in the ischemic cortex [F_(1,7), 23.30; P < 0.05] and striatum [F_(1,7), 13.92; P < 0.05], there was a strong interaction effect of ischemia and hormone treatment in both regions [F_(1,7), 22.39; P < 0.05; F_(1,7), 14.65; P < 0.05, respectively], indicating that hormone treatment inhibited ischemia-induced IGF-I up-regulation. Thus, IGF-I levels in the ischemic brain regions of hormone-treated animals were no different from their nonischemic counterparts.

Fig. 8. — Effect of vitamin D₃ injections on plasma and brain IGF-I levels. Plasma and brain tissue derived from control and vitamin D-treated female rats 5 d after stroke were analyzed for IGF-I expression. A, Vitamin D treatments significantly increased circulating IGF-I levels (39.5%) as compared with levels in control rats injected with vehicle alone (*, P < 0.05). *Bars* represent mean ± sem. B, IGF-I levels were elevated in the ischemic cortex and striatum, but this was only seen in the controls and not the vitamin D₃-treated animals. Thus, IGF-I levels were significantly lower in the ischemic cortex (11.5-fold) and striatum (3.4-fold) of hormone-treated animals. Data were analyzed by two-way ANOVA for hormone treatment and ischemia for each brain region; a, Main effect of hormone treatment; b, main effect of ischemia; c, interaction effect of hormone and ischemia. Group differences were considered significant at P < 0.05. *Bars* represent mean ± sem; n = 4–5 in each group.

Effects of acute vitamin D treatments on the central ischemic inflammatory response

In both vitamin D-treated and control animals, MCAo increased overall cytokine expression in the cortex and striatum of the ischemic hemisphere as compared with the nonischemic hemisphere. However, baseline cytokine expression was generally higher in vitamin D-treated females as compared with controls. This pattern contrasts with findings in VDD vs. control animals, wherein baseline cytokine expression was generally decreased in brain tissue from VDD animals. Vitamin D-treated females had significantly elevated cortical IL-2 and IL-5 (92.4% and 6.6-fold, respectively) and striatal IL-1β expression (22.1%) as compared with controls (P < 0.05) (Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). A trend (P = 0.06) was also found for increased IL-1β expression (45.6%) in cortical tissue from vitamin D-treated animals.

Discussion

The present data indicate that VDD adversely affects stroke outcome, whereas poststroke vitamin D supplementation was ineffective. The VDD diet effectively reduced circulating vitamin D₃ precursor (25-OHD) and active (1,25-OH₂D₃) forms of vitamin D compared with control chow. Reduced circulating 1,25-OH₂D indicates an endocrine mechanism of biologically active vitamin D function in stroke injury. Levels of 25-OHD were also lower in brain tissue of VDD animals in the acute stroke phase, and the tissue availability of both 25-OHD and 1,25-OH2D was further compromised by elevated expression of the vitamin D catabolic enzyme, CYP24A1, in the same tissue at 5 d after stroke. Thus, dietary vitamin D restriction likely constrains availability and function of vitamin D metabolites. At 5 d after MCAo, VDD groups showed greater cortical and striatal infarction, whereas behavioral impairment was observed as early as 3 d after stroke. Circulating and local ischemia-induced IGF-I levels were also reduced in VDD groups at 5 d after stroke. Additionally, VDD suppressed antiinflammatory cytokines associated with T-helper cells and macrophages in the ischemic brain. Vitamin D₃ injections significantly elevated circulating 25-OHD levels compared with controls. However, this treatment did not ameliorate infarct volume or behavioral deficits.

A growing body of data indicates that vitamin D levels are significantly lower in acute stroke patients vs. controls (69) and that low vitamin D levels increase the risk for future strokes (24) and are independently predictive for fatal strokes (28). Vitamin D is thought to modify the risk of CVD indirectly through chronic and inflammatory CV risk factors, such as diabetes, dyslipidemia, and metabolic syndrome (2, 14, 23, 70–72). Severely low vitamin D levels are reported in specific populations at risk for stroke, such as the elderly and nonwhite races (especially African Americans), whereas moderate deficiency is additionally seen in young and middle aged populations (25, 31, 73). The low levels of vitamin D in our VDD groups is comparable with those seen in stroke-prone human populations, and these low levels directly affect both brain tissue loss and exacerbate behavioral impairment. Furthermore, our data are also consistent with studies from other labs that report a neurotoxic role for VDD (59).

There are several mechanisms by which VDD could exacerbate stroke injury. VDD affects poststroke inflammatory responses, which play a critical role in the pathophysiology of cerebral ischemia (74–77). Cerebral ischemia results in a characteristic poststroke inflammatory response, involving activation of astrocytes and immune cells, increased vascular and blood brain barrier (BBB) permeability, influx of leukocytes, and cytokine production (38, 78, 79). Local immune cells (microglia) are activated initially, and subsequent peripheral immune cells gain access to the CNS as a result of a compromised BBB and increased adhesion molecule expression on cerebral vasculature and activated immune cells (78, 80, 81). Once activated, inflammatory cells can secrete cytotoxic substances, such as additional cytokines, that induce secondary damage and propagate immune cell activation and recruitment to the ischemic site (79, 81, 82). Although the poststroke inflammatory response is necessary to promote repair and regeneration (74, 83), infiltration of certain T-cell subsets may promote brain injury rather than facilitate recovery, whereas others are thought to provide neuroprotective benefits (74, 81, 84, 85). Additional cytokines released from recruited T cells at the ischemic site may play a key role in the pathogenesis of stroke, and the ratio of pro- to antiinflammatory T-cell subtypes is proposed to determine whether the poststroke inflammatory response will promote recovery or neurodegeneration. Although the adaptive immune response is generally suppressed by 1,25-OH₂D₃, the active form of vitamin D, this hormone nevertheless exerts direct effects on activation, phenotypic determinants, and secretions of T cells and antigen presenting cells (5, 86). Vitamin D enhances antiinflammatory Treg and Th2 development and cytokine production, whereas proinflammatory Th1 and Th17 subsets are suppressed (5, 87–90), suggesting a possible mechanism by which VDD may exacerbate ischemic cell loss. Although vitamin D has been shown to act as a potent immunomodulator capable of regulating cytokine secretion (2, 91–94), little is known of the effects of this hormone on inflammation in the context of ischemic stroke. The present study shows that IL-6 is specifically elevated in the ischemic brain of VDD animals. IL-6 and TGF-β have been shown to play an important role in regulating the Th17:Treg balance, and elevated IL-6 production inhibits Treg generation (95, 96). Our data and reports from other labs and clinical studies indicate that VDD promotes IL-6 expression (59, 97–99). We predict that VDD, directly or via regulation of cytokine intermediaries like IL-6 and TGF-β1, promotes an imbalance in T-cell subpopulations in the ischemic brain, which results in neurotoxicity.

The local inflammatory response in the brain is known to contribute to ischemic injury, yet the peripheral inflammatory response to stroke is only now becoming associated with neurological outcome (67, 77). Although antiinflammatory cytokine expression in brain tissue was reduced in VDD animals, cytokine levels in VDD splenic tissues were elevated vs. control levels, suggesting differential regulation between the central and peripheral components of the inflammatory response. The peripheral inflammatory response precedes brain inflammation in experimental stroke models and is characterized in part by increased IL-6, an established predictor of poor stroke outcome in humans (75, 77, 78). In vivo, depletion of macrophages from peripheral lymphoid organs before experimental stroke resulted in significant reduction of hematogenous (peripheral) macrophages at the ischemic site (66). Vitamin D is known to enhance innate immune responses, and microglia/macrophages mediate the intrinsic inflammatory response in the brain (66). Our experiments support vitamin D's role in both the central and peripheral ischemic inflammatory response.

In addition to its role as an immune modulator, VDD may also exacerbate ischemic cell loss by decreasing the availability of neuroprotective growth factors. Growth factors such as IGF-I contribute to CNS repair by promoting axon and dendrite sprouting and regrowth after stroke, which is a critical component of recovery of function (51), and also support the growth and survival of neural progenitor cells and other support cells, such as astrocytes and microglia, that play an integral role in poststroke recovery (52). Levels of IGF-I, a known vascular and neuroprotectant up-regulated in ischemia (100, 101), are positively correlated with vitamin D levels (49) and negatively correlated with ischemic stroke outcome in experimental and clinical studies (101–104). Furthermore, VDD is clinically associated with dysfunction or failure of the insulin/IGF-I axis in CVD states, such as diabetes and metabolic syndrome (49, 105, 106). Although IGF-I was up-regulated in the ischemic brain, VDD attenuated this peptide's expression, suggesting another mechanism by which VDD could exacerbate ischemic cell loss, because IGF-I provides support for local regrowth and repair mechanisms after stroke. Indeed, VDD animals had significantly lower IGF-I levels in ischemic tissues and also displayed more severe poststroke behavioral deficits. Although the liver is the single largest source, IGF-I is synthesized by diverse organs, including the brain (107), and exogenous IGF-I reduces ischemic injury in many species (103, 108–110). IGF-I has been shown to stimulate stroke-induced neurogenesis (111) and promote neuronal survival, neuronal myelination, and angiogenesis (112, 113). In stroke patients, lower levels of circulating IGF-I were predictive of a poorer outcome (114, 115, but see Ref. 116) and increased mortality (102, 117). Experimental studies have shown reciprocal regulation of the IGF-I and vitamin D systems. IGF-I promotes the hydroxylation of 25-OHD₃ into the active 1,25-OH₂D₃ hormone by inducing renal CYP27B1, the enzyme responsible for vitamin D activation (118). Our results support such a role for IGF-I, because levels of this neuroprotective peptide were reduced in VDD serum, liver, and brain tissue, whereas circulating 1,25-OH₂D₃ was significantly reduced in VDD animals. In the present study, systemic vitamin D injections increased circulating (plasma) IGF-I levels but did not attenuate ischemic brain injury. However, this treatment did not elevate IGF-I in ischemic tissues, suggesting that local IGF-I elevation may be critical to attenuate ischemic injury in female rats.

Poststroke brain IGF-I levels represent a complex mix of locally synthesized IGF-I and peripherally derived peptide, which is largely synthesized by the liver. In normal conditions, very little IGF-I crosses the BBB, but after stroke (and concomitant changes in the BBB), systemic IGF-I enters the brain more easily. In the VDD experiments, for example, brain levels of IGF-I mRNA increase on the infarcted side but are no different between control and VDD groups, indicating the reduced IGF-I protein levels in the VDD group reflects differences in circulating IGF-I influx after stroke. Curiously, we did not observe elevated brain IGF-I levels in the vitamin D-treated stroke animals, and one possible explanation for this may be linked to vitamin D's well-known effects on elevating IGF-binding proteins (118, 119), which are thought to protect IGF-I from proteolytic cleavage but may also inhibit transfer of peptide across the BBB. Alternatively, interactions between VDR and the IGF-binding proteins have been reported (120), and in many cases, such binding suppresses VDR-mediated signaling (121) and may indirectly account for the absence of IGF-related neuroprotection in vitamin D-treated and/or VDD groups.

Although VDD exacerbated ischemic damage, supraphysiologic vitamin D treatment did not ameliorate injury in our experiments. This may be due to the fact that the dose was suboptimal or that the timing of vitamin D treatment was not effective. The 10 μg/kg dose of vitamin D was chosen in an effort to overcome early-stage inflammatory pathology already in place 4 h after ischemia (78). The majority of studies demonstrating neuroprotection by vitamin D exclusively focused on preemptive vitamin D administration (15, 54, 122), which does not adequately address the clinical reality that most stroke patients only receive medical attention several hours after stroke. Thus, in the present studies, animals were injected with vitamin D 4 h after ischemia, which did not promote neuroprotection. A more likely possibility is that vitamin D may act synergistically with other neuroprotectants but not independently (123). For example, acutely administered vitamin D in combination with progesterone, a neuroprotective hormone, reduced brain injury in female rats, where vitamin D alone was ineffective (59).

Collectively, the present studies indicate that VDD is an independent modulator of stroke severity. Our data also support the hypothesis that VDD-induced stroke severity may be due to the suppression of endogenous neuroprotectants, such as IGF-I, as well as dysregulation of the inflammatory response after ischemia. Besides its role as a neuroprotectant, IGF-I may also shape the immune environment of the ischemic brain. IGF-I exerts proliferative effects on T cells (124), and IGF-I signaling influences the antiproliferative action of IFN-γ on polarized Th1 cells (125). Thus, one intriguing possibility is that VDD in combination with reduced IGF-I expression may alter the ratio of cytotoxic (Th1, Th17) to cytoprotective (Th2, Treg) T-cell cohorts and thus increase tissue damage.

Supplementary Material

Supplemental Data

supp_153_5_2420__index.html^{(912B, html)}

Acknowledgments

This work was supported by National Institutes of Health grants AG027684 and AG028303 (to F.S.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:

BBB: Blood brain barrier
CNS: central nervous system
CVD: cardiovascular disease
CYP24A1: 24-hydroxylase
CYP27B1: 1-α-hydroxylase
ET-1: endothelin-1
MCAo: middle cerebral artery occlusion
25-OHD: 25-hydroxyvitamin D
TTC: 2,3,5-triphenyl-tetrazolium chloride
VDD: vitamin D deficiency
VDR: vitamin D receptor.

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PERMALINK

Vitamin D Deficiency Exacerbates Experimental Stroke Injury and Dysregulates Ischemia-Induced Inflammation in Adult Rats

Robyn Balden

Amutha Selvamani

Farida Sohrabji

Abstract

Materials and Methods

Animals

Vitamin D treatment

VDD studies

Vitamin D treatment studies

Middle cerebral artery occlusion

Sensorimotor tests

Tape test

Infarct volume

Tissue collection

Vitamin D levels

IGF-I levels

Quantitative RT-PCR

Cytokine assay

TGF-β1 levels

Statistical analysis

Results

VDD diet studies

Weight analysis

Local and circulating levels of 1,25-OH2 vitamin D and 25-OH vitamin D

Fig. 1.

Local expression of vitamin D-associated genes

Infarct volume

Fig. 2.

Behavioral outcomes

Fig. 3.

Local and circulating levels of IGF-I

Fig. 4.

Effects of VDD on the central ischemic inflammatory response

Fig. 5.

Effects of VDD on the peripheral ischemic inflammatory response

Acute vitamin D treatment studies

Fig. 6.

Effect of acute vitamin D3 treatment on infarct volume.

Fig. 7.

Behavioral analysis

Local and circulating levels of IGF-I

Fig. 8.

Effects of acute vitamin D treatments on the central ischemic inflammatory response

Discussion

Supplementary Material

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Local and circulating levels of 1,25-OH₂ vitamin D and 25-OH vitamin D

Effect of acute vitamin D₃ treatment on infarct volume.