Magnesium supplement promotes sciatic nerve regeneration and down-regulates inflammatory response

Abstract

Magnesium (Mg) supplements have been shown to significantly improve functional recovery in various neurological disorders. The essential benefits of Mg supplementation in peripheral nerve disorders have not been elucidated yet. The effect and mechanism of Mg supplementation on a sciatic nerve crush injury model was investigated. Sciatic nerve injury was induced in mice by crushing the left sciatic nerve. Mice were randomly divided into three groups with low-, basal- or high-Mg diets (corresponding to 10, 100 or 200% Mg of the basal diet). Neurobehavioral, electrophysiological and regeneration marker studies were conducted to explore nerve regeneration. First, a high Mg diet significantly increased plasma and nerve tissue Mg concentrations. In addition, Mg supplementation improved neurobehavioral, electrophysiological functions, enhanced regeneration marker, and reduced deposits of inflammatory cells as well as expression of inflammatory cytokines. Furthermore, reduced Schwann cell apoptosis was in line with the significant expression of bcl-2, bcl-X(L) and down-regulated expression of active caspase-3 and cytochrome C. In summary, improved neurological function recovery and enhanced nerve regeneration were found in mice with a sciatic nerve injury that were fed a high- Mg diet, and Schwann cells may have been rescued from apoptosis by the suppression of inflammatory responses.

Full text

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium Research 2011; 24 (2): 54-70 ORIGINAL ARTICLE
Magnesium supplement promotes
sciatic nerve regeneration and
down-regulates inflammatory
response
Hung-Chuan Pan1,6, Meei-Ling Sheu3, Hong-Lin Su4, Ying-Ju Chen2, Chun-Jung
Chen2, Dar-Yu Yang5, Wen-Ta Chiu6, Fu-Chou Cheng2
1Department of Neurosurgery; 2Stem Cell Center, Department of Medical Research, Taichung
Veterans General Hospital, Taichung; 3Institute of Biomedical Sciences; 4Institute of Life
Sciences, National Chung-Hsing University, Taichung; 5Department of Neurosurgery, Chang
Bing Show Chwan Memorial Hospital, Changhua; 6Department of Neurosurgery, Taipei
Medical University, Shuang Ho Hospital, Taipei, Taiwan
Correspondence: Dr Fu-Chou Cheng, Stem Cell Center, Department of Medical Research, Taichung Veterans
General Hospital, No. 160, Taichung Harbor Rd., Sec. 3, Taichung 40705, Taiwan
<vc1035@gmail.com>
Abstract. Magnesium (Mg) supplements have been shown to significantly
improve functional recovery in various neurological disorders. The essential
benefits of Mg supplementation in peripheral nerve disorders have not been
elucidated yet. The effect and mechanism of Mg supplementation on a sciatic
nerve crush injury model was investigated. Sciatic nerve injury was induced in
mice by crushing the left sciatic nerve. Mice were randomly divided into three
groups with low-, basal- or high-Mg diets (corresponding to 10, 100 or 200% Mg of
the basal diet). Neurobehavioral, electrophysiological and regeneration marker
studies were conducted to explore nerve regeneration. First, a high Mg diet sig-
nificantly increased plasma and nerve tissue Mg concentrations. In addition,
Mg supplementation improved neurobehavioral, electrophysiological functions,
enhanced regeneration marker, and reduced deposits of inflammatory cells as
well as expression of inflammatory cytokines. Furthermore, reduced Schwann
cell apoptosis was in line with the significant expression of bcl-2, bcl-XLand
down-regulated expression of active caspase-3 and cytochrome C. In summary,
improved neurological function recovery and enhanced nerve regeneration were
found in mice with a sciatic nerve injury that were fed a high- Mg diet, and
Schwann cells may have been rescued from apoptosis by the suppression of
inflammatory responses.
Key words: cytokines, inflammation, magnesium, nerve regeneration, sciatic nerve
injury
Neural trauma has been shown to result in
endoneurial production of cytokines and their
mRNA, including interleukin (IL)-1␤, tumor
necrosing factor (TNF)-␣, IL-6, and Interferon
(INF)-␥[1-4]. The production of TNF-␣and IL-1
following nerve injury is a critical determinant
of later sequelae of injury and inflammation in
peripheral nerves, particularly because of the key
roles of these cytokines in Wallerian degeneration
(WD) [1, 2, 5, 6]. TNF-␣and IL-1 are of particu-
lar significance to WD. First, they can initiate the
cytokine networks of WD as they do in other net-
works of inflammation. Second, they contribute
to macrophage recruitment to inflammatory sites
through endothelial cell activation and chemokine
production as well as myelin removal [7].
54
doi:10.1684/mrh.2011.0280
To cite this article: PanHC, Sheu ML, Su HL, Chen YJ, Chen CJ, Yang DY, Chiu WT, Cheng FC. Magnesium supplement promotes
sciatic nerve regeneration and down-regulates inflammatory response. Magnes Res 2011; 24(2): 54-70 doi:10.1684/mrh.2011.0280

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium promotes nerve regeneration and regulates inflammation
Furthermore, they can indirectly regulate sur-
vival and growth of peripheral nervous system
neurons and neuropathic pain through the regula-
tion of nerve growth factor (NGF) production [8, 9].
Nerve tissue injury in general leads to a
local inflammatory response that is regulated by
numerous signal molecules, including cytokines.
Their production and local effects may also affect
the outcome of nerve trauma. Cytokines act in
a network-like fashion and are crucial in nerve
regeneration. However, TNF-␣has been previ-
ously described as a potent cytotoxic molecule
during active phases of demyelination in vari-
ous forms of peripheral neuropathy [10]. In
addition, exogenous administration of TNF-␣
increased Schwann cell apoptosis and viability in
axotomized mice [11, 12]. In our previous inves-
tigation, we found that attenuation of TNF-␣
in a sciatic nerve crush injury model prevented
Schwann cell apoptosis and further escalated
nerve myelination [3]. Furthermore, the partly
abolished expression of inflammatory cytokines
such as TNF-␣, Il-1␤, and INF-␥contributed to a
decrease in Schwann cell apoptosis with a subse-
quent increase in nerve regeneration [4, 13]. Thus,
the intensity of responsive cytokine expression in
peripheral nerve injury modulated WD, attenu-
ated nerve regeneration, and played a cytotoxic
role in orchestrating Schwann cell apoptosis. The
optimal elicitation of the appropriate expression
of cytokines should be finely tuned to allow for
removal of myelin debris without exacerbation of
the property that is cytotoxic to Schwann cells.
It is known that magnesium (Mg) is crucial
for cellular bioenergetics, DNA synthesis, RNA
aggregation, protein synthesis, the functioning
of ATPase, and plasma membrane integrity. Mg
treatment blocks the mitochondrial permeability
transition pore, calpain activation, lipid peroxida-
tion, and the production of oxygen-free radicals
[14]. The first observation of clinical symptoms of
inflammation in Mg-deficient rats was published
in the 1930s [15]. Significant elevation of plasma
levels of several cytokines and substance P was
observed in rats after 3 weeks of Mg deficiency
[16]. Furthermore, a rise in plasma concentration
of IL-6 was also found in rats that were fed an
Mg-deficient diet [17]. The stress-induced over-
production of TNF-␣appears as early as 2 days
after initiation of Mg-deficient diets [18]. In addi-
tion, Mg deficiency upregulates IL-1␣and IL-6,
and pleiotropic cytokines have been implicated
in acute phase response and inflammation [19].
Experimental Mg deficiency in the rat has been
shown to induce a clinical inflammatory syndrome
characterized by leukocyte and macrophage acti-
vation, release of inflammatory cytokines and
acute phase proteins, and excessive production of
free radicals. The priming of phagocytic cells, the
opening of the calcium channel, the activation of
N-methyl-D-aspartate (NMDA) receptors, and the
activation of nuclear factor-kappa B (NF␬B) have
been considered as potential mechanisms [20].
Mg deficiency contributes to an exagger-
ated response to immune stress and oxida-
tive stress as a consequence of the inflam-
matory response. Inflammation contributes to
the pre-atherosclerotic changes in lipoprotein
metabolism, endothelial dysfunction, thrombosis,
hypertension and the development of metabolic
syndromes [20]. Although suppression of the
inflammatory response has been shown to rescue
Schwann cells from apoptosis to promote nerve
regeneration [3, 4, 13], there are insufficient data
to support the hypothesis that down-regulation
of the inflammatory response by the administra-
tion of Mg is highly correlated with a protective
effect on peripheral nerve injury. To date, except
for a few reports that address the administra-
tion of Mg supplements to prevent motor neuron
death in neonatal sciatic nerve injury [21], there
are scant data concerning the effects of either Mg
supplements or deficiency on functional recovery
in adult sciatic nerve injury, especially in regard
to the relationship of inflammatory response and
nerve regeneration. In this study, three groups
of animals were given diets supplemented with
basal-, low- or high-dose Mg and actual concen-
trations of Mg in plasma and various tissues
were then determined. Subsequently, these three
groups of animals were subjected to sciatic nerve
crush injury and then given the same respec-
tive Mg-supplemented diets (to determine the
relationship of nerve regeneration with inflam-
matory response). Furthermore, the molecular
mechanism contributing to these effects was also
investigated.
Material and methods
Crush models
Imprinting control region (ICR) mice weighing
30-40 g were used in this study. Permission was
obtained from the Ethics Committee of Taichung
55

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
H. C. PAN ET AL.
Veterans General Hospital for their use. Before
the animals were subjected to the crush injury,
they were divided into three groups and fed a
basal diet (5755 TestDiet containing 0.7 mg/g Mg,
Richmond, IN, USA), a low-Mg diet (containing
<0.08 mg/g Mg, 5865 TestDiet), or a high-Mg
diet (5755 TestDiet containing 0.7 mg/g Mg and
supplemented by MgCl2-0.5 mg/mL in water) for
3 weeks before experiments and 4 weeks after
sciatic nerve injury. The mice were anesthetized
with 2% isoflurane during induction followed by a
maintenance dose (0.5-1%). The left sciatic nerve
was exposed under a microscope using the gluteal
muscle splitting method. A vessel clamp (B-3,
pressure 1.5 gm/ mm2, S&T Marketing, Ltd.,
Switzerland) was applied 10 mm from the inter-
nal obturator canal for 20 minutes, as previously
described [22]. The crush site was sutured with
9-0 nylon over the epineuria as the mark. After
injury, the animals were fed the same respective
diets for another 4 weeks. For determination of
the Mg concentrations in plasma and various
tissues, six animals in each group were used. To
assess the Mg concentration in plasma after crush
injury, blood was withdrawn from six animals
per group at one-week intervals for four weeks.
Neurobehavior, electrophysiology, and histology
were assessed in six animals in each group. At
three different time points, six animals per group
(a total of 18 animals in each group) were used for
determination of inflammatory cell infiltration
and associated gene expression. Six animals per
group were used for determination of inflamma-
tory cytokine levels and blood was withdrawn
for determination of activity of macrophages and
neutrophils. At three different time points, six
animals per group (a total of 18 animals in each
group) were used for TUNEL assay. At four differ-
ent time points, six animals per group (a total of
24 animals in each group) were used for analysis
of anti-apoptotic associated gene expression.
Various tissues (sciatic nerve, spinal cord, brain,
muscle and small intestine) were harvested
and Mg concentrations were determined four
weeks after injury. Mg concentrations in blood
and tissues were determined using a biochemical
analyzer (TBA-200 FR, Toshiba Medical Products,
Tokyo, Japan).
Analysis of functional recovery
A technical assistant who was blinded to treat-
ment allocation evaluated sciatic nerve function 4
weeks after the surgery. The evaluation method
included ankle kinematics [23] and sciatic func-
tion index (SFI) [24]. In the sagittal plane
analysis, the following formula was used in the
mechanical analysis of the rat ankle: ␪ankle=␪
foot- leg 90. Several measurements were taken
from the footprint: (i) distance from the heel to
the third toe, the print length (PL); (ii) distance
from the first to the fifth toe, the toe spread (TS);
and (iii) distance from the second to the fourth
toe, the intermediary toe spread (ITS). All three
measurements were taken from the experimental
(E) and normal (N) sides. The SFI was calculated
according to the equation:
SFI=38.3(EPL-NPL/NPL)+109.5(ETS-NTS/
NTS)+13.3(EIT-NIT/NIT)-8.8
The SFI oscillates around 0 for normal nerve
function, whereas SFI around -100 represents
total dysfunction [24].
Electrophysiological study
Sciatic nerves from individual groups were
exposed 4 weeks after operation, and electrical
stimulation was applied to the proximal side of
the injured site; the evoked compound muscle
action potential (CMAP) amplitudes and conduc-
tion latency were recorded in the gastrocnemius
with an active monopolar needle electrode 10 mm
below the tibia tubercle and with a reference nee-
dle 20 mm from the active electrode. The stimu-
lation intensity and filtration ranges were 2.5 mA
and 20-2,000 Hz, respectively. A similar assess-
ment was performed on the non-injured side.
The CMAP data and conduction latency were
converted to the ratio of the injured side divided
by the normal side to adjust for the effect of
anesthesia [22].
Western blot
The nerve tissue (10 mm) (9-0 nylon marker in
the middle of the nerve) was harvested after treat-
ment at different time points and proteins were
extracted. Proteins (50 ␮g) were resolved by SDS-
polyacrylamide gel electrophoresis and trans-
ferred onto a blotting membrane. After blocking
with non-fat milk, the membranes were incubated
with antibodies against bcl-2 (anti-mouse; 1:1,000
dilution; BD Biosciences, Franklin Lakes, NJ,
USA), bcl-XL(anti-rabbit; 1:1,000 dilution; Santa
Cruz Biotechnology, Santa Cruz, CA, USA), active
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Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium promotes nerve regeneration and regulates inflammation
caspase-3 (anti-mouse; 1:1,000 dilution; Abcam,
Cambridge, MA, USA), cytochrome C (anti-rabbit;
1:1,000 dilution; Santa Cruz Biotechnology), Bad
(anti-mouse; 1:1,000 dilution; Santa Cruz Biotech-
nology), Bax (anti-rabbit; 1:1,000 dilution; Santa
Cruz Biotechnology), and ␤tubulin (anti-mouse;
1:10,000 dilution; Santa Cruz Biotechnology)
overnight at 4◦C. The membranes were incu-
bated with horseradish peroxidase-conjugated
secondary antibody and developed using ECL
Western blotting reagents. The intensity of pro-
tein bands was determined using a computer
image analysis system (IS1000, Alpha Innotech
Corporation, Santa Clara, CA, USA).
Isolation of RNA and reverse
transcriptase-polymerase chain reaction
(RT-PCR)
The isolation of RNA, synthesis of cDNA, and PCR
were carried out as previously described [4]. DNA
fragments of specific genes and internal controls
were co-amplified in a single one-step RT-PCR set-
up. The PCR reaction was carried out under the
following condition: one cycle of 94◦C for 3 min, 28
cycles of (94◦C for 50 s, 58◦C for 40 s, and 72◦C for
45 s), and then 72◦C for 5 min. The amplified DNA
fragments were resolved by 1.5% agarose gel elec-
trophoresis and stained with ethidium bromide.
The intensity of each signal was determined using
a computer image analysis system (IS1000; Alpha
Innotech Corporation). The primer sets used in the
study were: 5’-CACCGTCATCCTCGTTGC and 5’-
CACTTGGCGGTTCTTTCG for RANTES; 5’-CAG
GTCTCTGTCACGCTTCT and 5’-AGTATTCATG
GAAGGGAATAG for MCP-1; and 5’-TCCTGTGG
CATCCATGAAACT and 5’-GGAGCAATGATCT
TGATCTTC for ␤-actin (as a reference gene).
␤-actin control was analyzed per plate of exper-
imental gene to avoid plate-to-plate variation.
Final RT-PCR data are expressed as the ratio
of copy numbers of experimental gene per 103or
104copies of ␤-actin for samples performed in
duplicates.
Quantification of pro-inflammatory
cytokines
Six nerve tissues in each group for each sin-
gle parameter were removed 7 days after sciatic
injury. These tissues (10 mm in length) were
retrieved and stored at -80◦C. Subsequently, each
tissue sample was homogenized with Laemmli
SDS buffer. The homogenate was centrifuged
for 10 minutes at 12,000 g at 4◦C. The tissue
homogenate, 100 ␮L in triplicate, was applied to a
microtiter plate and allowed to adhere overnight
at 4◦C. The microtiter plates were washed with
phosphate-buffered saline (PBS)-Tween-20 and
blocked with 1% BSA in PBS (200 ␮L) for 1 h. The
plates were then treated with respective primary
antibodies and allowed to incubate for 6 hours at
37◦C. One hundred ␮L of the respective polyclonal
antibodies against TNF-␣(anti-goat; 1:100 dilu-
tion; R&D Systems, Inc., Minneapolis, MN, USA),
IL-1␤(anti-goat; 1:200 dilution; R&D Systems,
Inc.), IL-6 (anti-goat; 1:300 dilution, R&D Sys-
tems, Inc.) and INF-␥(anti-goat; 1:150 dilution,
Chemicon, Inc., Billerica, MA, USA) were applied
overnight to microtiter plates. After further wash-
ing in PBS-Tween-20, the plates were incubated
with the respective second antibody conjugated to
alkaline phosphate (100 ␮L) for 1 h. The reaction
was developed using p-nitrophenyl phosphate,
disodium (3 mM) in carbonate buffer, pH 9.6 [100
mM Na2CO3and 5 mM MgCl2(150 ␮L)], and the
reaction was terminated after 30 minutes using
0.5 N NaOH (50 ␮L). The absorbance of colored
product was read at 450 nm using a microplate
reader (Bio-Tek Instruments, Winooski, VT, USA).
The relative amount of antigen present was mea-
sured from the densitometric reading against a
standard curve.
Neutrophil and monocyte/macrophage
isolation
Blood was collected from the abdominal aorta
of pentobarbital-anesthetized mice 7 days after
operation. Neutrophils and monocytes/macro-
phages were purified by dextran sedimentation,
centrifugation through Ficoll-Hypaque, and hypo-
tonic lysis of erythrocytes. Cell migration was
evaluated with a modified 24-well Transwell, as
previously described [25]. Cells (1 ×106) were
added to the upper well of the chamber. The
lower well received RPMI containing 10% fetal
bovine serum. The upper and lower wells were
separated by 3-␮m pore size polycarbonate filters,
and the chamber was incubated for1hat37
◦C.
Migrating cells attached to the lower surfaces
were fixed in ethanol for 10 min and stained with
crystal violet. Labeled cells were counted micro-
scopically at 400 x magnification within a total
57

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H. C. PAN ET AL.
area of 2 mm2. For IL-1␤analysis, the obtained
monocytes/macrophages were incubated with
RPMI alone or stimulated with lipopolysaccha-
ride (LPS) (10 ng/mL)/interferon-gamma (IFN-␥)
for 24 h. The supernatants were collected and
subjected to ELISA for the measurement of IL-1␤.
Terminal deoxynucleotidyl
transferase-mediated biotinylated UTP
nick-end labeling (TUNEL) assay
The nerve tissue (10 mm) obtained after crush
injury was embedded, cut longitudinally into
8␮m-thick sections and subjected to TUNEL
assay (Roche Molecular Biochemicals, Mannheim,
Germany) according to the manufacturer’s
instructions 3, 7, and 14 days after injury. Apop-
totic cells were defined as those cells that were
TUNEL and S-100 positive. Among longitudinal
consecutive resections, five consecutive resections
contiguous to a maximum diameter were chosen
to be measured. Of 100 squares with a surface
area of 0.01 mm2each, 20 squares were randomly
selected in an ocular grid and used to count
the number of cells. The number of apoptotic
transplanted cells was expressed as a percentage
of the total number of nuclei counted, with at
least 25,000 nuclei for each count [26].
Immunohistochemistry study
Serial 8 ␮m-thick sections of sciatic nerve were cut
on a cryostat, mounted on superfrost/plus slides
(Menzel-Glaser, Braunschweig, Germany) and
subjected to immunohistochemistry with antibod-
ies against CD68, CD 34 (Chemicon, 1:200 dilu-
tion), CD 8, CD3 (Serotec, Dusseldorf, Germany;
1:200 dilution), CD19 (Thermo, Waltham, MA,
USA; 1:200 dilution), neutrophil (Abcam, 1:200
dilution), neurofilament (Chemicon, 1:300 dilu-
tion), and S-100 for detection of inflammatory
cells and nerve fibers, respectively. The immuno-
reactive signals were observed using goat anti-
mouse IgG (FITC) (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA, USA ; 1:200
dilution), anti-mouse IgG (Rhodamine) (Jackson,
1:200 dilution), or 3, 3’-diaminobenzidine brown
color. Among longitudinal consecutive resections,
five consecutive resections contiguous to a maxi-
mum diameter were chosen to be measured. Of
100 squares with a surface area of 0.01 mm2each,
20 squares were randomly selected in an ocular
grid and used to count the number of cells.
For the determination of neurofilament, six nerves
in each group were cut longitudinally into 8 ␮m-
thick sections, and stained with each antibody.
The region of maximum diameter of the resected
nerve tissue with crush mark was chosen to be
examined. Areas of activities (0.2 mm2) appeared
dense against background and were measured
using a computer image analysis system (Alpha
Innotech Corporation, IS1000).
Histological examination
After neurobehavioral and electrophysiological
testing, six mice in each group underwent tran-
scardial perfusion with 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) after being re-
anesthetized. The bilateral gastrocnemius muscle
from the bones was sent for measurement of
muscle weight. The nerve was embedded, cut
longitudinally into sections 8 ␮m thick and
stained with haematoxylin-eosin (H&E) for the
measurement of vacuole number and S-100 for
the determination of myelination. The methods
for determining the numbers of vacuoles and
density against S-100 have been described pre-
viously [26]. The left sciatic nerve was harvested
from the animals after electrophysiological test-
ing and the nerve tissue was fixed on a plastic
plate using stay suturing to keep the nerve
straight [27].
Statistical analysis
Data are expressed as mean±SE (standard error).
The statistical significance of differences between
groups was determined by one-way analysis of
variance (ANOVA) followed by Dunnett’s test. For
SFI, angle of ankle, and CatWalk analysis, the
results were analyzed by repeated-measurement
of ANOVA followed by Bonferroni’s multiple com-
parison method. A pvalue of less than 0.05 was
considered significant.
Results
Alteration of Mg concentrations in
plasma and various tissues
Approximately, each ICR mouse consumed
3 g/day and 5 mL/day water during this study.
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Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium promotes nerve regeneration and regulates inflammation
The estimated Mg daily dietary intakes of low-Mg
(ca. 0.24 mg/day), basal (ca. 2.1 mg/day), and
high-Mg (ca. 4.6 mg/day) diets of mice are shown
in table 1. Significant elevation of plasma Mg con-
centrations up to 1.51 mM were observed in mice
which received high Mg supplementation for 3
weeks. Depletion of Mg in the low Mg diet caused
low plasma Mg concentrations down to 0.48 mM
when compared to the control (0.92 mM). This
trend toward escalation or decrease in plasma
Mg concentrations was persistent after crush
injury (figure 1A). Mg concentrations in nerve
tissues were highly correlated with various Mg
diets. In other tissues such as spinal cord, brain,
muscle, and intestine, the same trend was also
demonstrated (figure 1B).
Neurobehavior study
High-dose Mg supplementation induced a signifi-
cant improvement in SFI compared with findings
obtained in the basal diet group (p<0.001), and
low-dose Mg supplementation further exacer-
bated SFI (p<0.001) (figure 2A). The angle of
ankle also showed a similar trend (figure 2B).
The ratios of CMAP (Lt/Rt) in the basal, low-
dose Mg, and high-dose Mg groups were 22±1.8%,
15±2.3%, and 49±3.2%, respectively (p<0.001).
The ratios of conduction latency in the basal,
low-dose Mg, and high-dose Mg groups were
3.05±0.19, 3.3±0.15, and 1.71±0.1, respectively
(p<0.001). The ratios of muscle weight in the
basal, low-dose Mg, and high-dose Mg groups
were 49±1.4%, 40±0.11%, and 66±1.2%, respec-
tively (p<0.001). Hence, the parameters of CMAP,
conduction latency, and muscle weight were
decreased by low-dose Mg supplementation and
then restored and augmented by a high-dose of
Mg (figure 2C).
Early and late nerve regeneration
augmented by Mg supplementation
High-dose Mg supplementation resulted in sig-
nificantly greater enhancement of neurofilament
expression (from 441.5±34.6 to 1115±24.8 rela-
tive density/mm2)(p<0.001), whereas low-dose
Mg supplementation abrogated the expression
to 211.3±12.1 relative density/mm2(p<0.001)
(figure 3A, B). The parameters of late nerve
regeneration were represented as the intensity of
myelination as evidenced by the increased expres-
sion of S-100 (from 561±28.9 to 903±30.4 rela-
tive density/mm2;p<0.001) after high-dose Mg
supplementation and decreased expression after
low-dose Mg supplementation (358±19.1 rela-
tive density/mm2)(p<0.001) (figure 3A, B).
Inflammatory response after Mg
supplementation
The immunohistochemical results in the
basal-dose Mg group showed an accumula-
tion of inflammatory cells starting at 3 days
(28.3±0.8/0.05mm2), reaching a plateau at 7 days
(32.3±1.1/0.05mm2)(p<0.001) and declining
at 14 days (19.2±0.6/0.05mm2)(p<0.001). The
administration of high-dose Mg supplements sup-
pressed the macrophage deposits (12±1/0.05mm2,
15±1.1/0.05mm2, and 11±1.5/0.05mm2)at3,7,
and 14 days, respectively (p<0.001). In contrast,
the administration of low-dose Mg supple-
ments stimulated the macrophage deposition
(36.2±0.94/0.05mm2, 47.8±2.1/0.05mm2, and
25.2±1.1/0.05mm2) at 3, 7, and 14 days, respec-
tively (p<0.001) (figure 4A, B). Significant
induction of MCP-1 and RANTES was observed
in groups treated with low-dose Mg supplemen-
tation. Furthermore, the alteration of MCP-1 and
RANTES was highly associated with deposits of
macrophages (figure 4C).
Table 1. Estimated total Mg intakes (mg/day) based on Mg contents in the diets and water in the three
studied groups.
Food intake
(g/day)
Mg in food
(according to
TestDiet)
(mg/day)
Water intake
(mL/day)
Mg in water
(mg/mL)
Estimated total
Mg intake
(mg/day)
Low Mg diet ∼3 0.24 ∼5 0 0.24
Basal Mg diet ∼3 2.1 ∼5 0 2.1
High Mg diet ∼3 2.1 ∼5 2.5* 4.6
*High Mg diet was supplemented by MgCl2in water (0.5 mg/mL).
59

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H. C. PAN ET AL.
Plasma Mg (mM)Mg (mmol/Kg wet weight)
4
3
2
1
0
4
3
2
1
0
-21 Crush 0
Sciatic
nerve
Spinal
cord
***
***
***
***
**
**
**
*
*
*
*
*
*
*
*
**
**
**
Brain Muscle Intestine
7142128Days
Basal Mg
Low Mg
High Mg
A
B
Figure 1. Plasma and tissue concentrations of Mg altered by various Mg diets.
A) Concentrations of plasma Mg at different time intervals.
B) Mg concentrations in various tissues at 4 weeks after sciatic injury.
N=6; ** p<0.01; *** p<0.001 (relative to basal Mg diet).
The elevated production of IL-1␤, IL-6, TNF-␣
and IFN-␥was decreased in the high-dose Mg
groups and further enhanced in the low-dose
Mg groups (figure 5A). To further explore the
inflammatory response in systematic circulation
following treatment with various doses of Mg,
neutrophils and monocytes/macrophages were
isolated from circulating blood 7 days after
crush injury. Monocytes/macrophages obtained
from injured animals treated with basal-dose Mg
supplements showed more elevated cell migra-
tion than those in the sham group. Up-regulated
cell migration was enhanced by low-dose Mg
supplements and inhibited by high-dose Mg
supplements. In contrast, neutrophils showed
similar levels of cell migration, but the level
60

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Magnesium promotes nerve regeneration and regulates inflammation
A0
-20
-40
-60
-80
-100
-120
Scores of SFI
0 7 14 21 28
Days
B
0
20
40
60
80
100
120
Angle of ankle
0 7 14 21 28
Days
Basal Mg
Low Mg
High Mg
*** ***
***
*
*
**
*
*
*
*
*
*
**
**
**
**
MW CAMP Latency
Ratio of Lt/ Rt
4
3.5
3
2.5
2
1.5
1
0.5
0
C
Basal Mg
Low Mg
High Mg
Basal Mg
Low Mg
High Mg
Figure 2. Illustration of neurobehavioral and electrophysiological studies.
A) Representative illustration of SFI and (B) angles of ankle at different time points for the three
studied Mg diets. C) Quantitative analysis of ratio (Lt/Rt) of muscle weight, CMAP, and conduction
latency.
Lt: left; Rt: right; SFI: sciatic nerve function index; MW: muscle weight; CMAP: compound muscle
action potential.
N=6; *p<0.05; ** p<0.01; *** p<0.001 (relative to basal Mg diet).
of expression was much weaker than that in
macrophages (figure 5B). Monocytes/macrophages
obtained from injured animals spontaneously
released higher levels of IL-1␤than the sham-
operated group. A high dose of Mg inhibited IL-1␤
release and a low dose enhanced the release
(figure 5C). After stimulation with LPS/IFN-
␥, the four groups of monocytes/macrophages
produced elevated levels of IL-1␤. The trend
toward IL-1␤release after stimulation was
similar to that in groups without stimulation
(figure 5D).
Cells rescued from apoptosis in crushed
nerve
Following nerve crush injury, a basal dose of
Mg administration induced Schwann cell apop-
tosis of 0.37±0.031% at 3 days, which reached
a plateau of 7.3±1.4% at 7 days (p<0.001)
and declined to 4.4±0.3% at 14 days (p<0.001).
High-dose Mg supplementation attenuated the
apoptosis to 0.1±0.01%, 3.01±0.27% (p<0.001),
and 1.4±0.11% (p<0.001) at 3, 7, and 14
days, respectively. Low-dose Mg supplementation
61

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
H. C. PAN ET AL.
B
A
Basal
Mg
Low
Mg
High
Mg
NF S-100
S-100
S-100
*
*
*
*
*
*
NF
NF
Basal Mg
Low Mg
High Mg
***
***
***
***
Relative density/mm2
1,200
1,000
800
600
400
200
0
NF S-100
Figure 3. Expression of nerve regeneration markers in the Mg diet groups.
A) Expression of neurofilament and S-100 at one and four weeks after injury treated with different Mg
doses.
B) Quantitative analysis of neurofilament and S-100.
N=6; *** p<0.001 (relative to basal Mg diet).
NF: neurofilament. Bar length=500 ␮m.
augmented the cell apoptosis to 6.6±0.24%
(p<0.001), 11.6±0.5% (p<0.001), and 5.9±0.24%
(p<0.001) at 3, 7, and 14 days, respectively
(figure 6A, B). In Western blotting analysis, Mg
depletion significantly enhanced Schwann cell
apoptosis by decreasing Bcl-2 and Bcl-XL expres-
sion levels, and subsequently increased active
caspase-3 and cytochrome C compared with those
of the control group. However, supplementation of
Mg also enhanced the expression of bcl-2 and bcl-
XLand decreased active caspase-3 and cytochrome
C when compared with values obtained in controls
(figure 7A, B).
Discussion
Various doses of Mg in the diet profoundly
influenced the alteration of Mg concentration in
plasma and tissues. In addition, Mg deficiency
markedly enhanced the inflammatory response,
and Mg supplementation counteracted the inflam-
matory response. Suppression of the inflamma-
tory response was highly associated with improve-
ment of neurological function, including neuro-
behavior and nerve myelination, whereas augme-
ntation of the inflammatory response was highly
associated with deterioration of neurological
62

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium promotes nerve regeneration and regulates inflammation
BC
A
3
60
50
40
30
20
10
0
25
20
15
10
5
0
14 Days MCP-1 RANTES
7
Basal
Mg
Low
Mg
High
Mg
Day 3 Day 7 Day 14
***
***
***
***
***
***
***
**
**
*
Intensity arbitary unit
CD 68(counts/0.5mm2)
Basal Mg
Low Mg
High Mg
Figure 4. Determination of inflammatory cell and associated cytokine levels. The nerve tissues were
retrieved at 3, 7, and 14 days after sciatic injury and were subjected to immunohistochemistry studies
with antibodies against CD68 and RT-PRC for determination of MCP-1 and RANTES at 7 days after
treatment with three Mg diets. A) Deposits of macrophages at different time points.
B) Quantitative analysis of inflammatory cells.
C) Quantitative analysis of MCP-1 and RANTES.
*p<0.05; ** p<0.01; *** p<0.001 (relative to basal Mg diet).
N=6. Bar length=100 ␮m.
function. The inflammatory response in the
injured nerve paralleled the increased cell apopto-
sis and immune cell deposits. The phenomenon of
reduced cell apoptosis was in line with increased
expression of bcl-2 and bcl-XLand was found to
be inversely correlated with expression of active
caspase-3 and cytochrome C. Thus, either high Mg
diet before and/or continuous supplementation of
Mg after sciatic injury may rescue cells from apop-
tosis by suppressing the inflammatory responses.
The anti-inflammatory effect was consistent with
the significant improvement in neurological func-
tion.
The alteration of plasma Mg in mice given vari-
ous Mg diets was similar to those found in previous
reports [28-30]. In addition, our data indicate that
these diets significantly influenced the concentra-
tions of Mg in plasma and tissues either 3 weeks
before or 4 weeks after sciatic nerve injury. How-
ever, it is difficult to compare these plasma and
tissue Mg concentrations among various species
of C57/BL6, ICR and other strains of mice. In the
rat model, SD rats are suitable for use in scia-
tic nerve injury models. In the present study, we
initially used C57/BL6 mice in our sciatic nerve
injury model as they are frequently used in the
63

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
H. C. PAN ET AL.
B
C
A
***
***
***
*** *** ***
**
**
*
***
**
***
**
***
*
*
*
*
*
*
Basal Mg
Low Mg
700 250
200
150
100
50
0
600
300
100
200
0
400
500
700
Monocyte/Macrophage
Neutrophil
TNF-αIL-1βINF-γIL-6 Basal Mg
(Sham)
Basal Mg Low Mg High Mg
Basal Mg
(Sham)
Basal Mg Low Mg High Mg
1L-1β (pg/mL)
Relative migration (%)
No stimulation
Stimulation
600
300
100
200
0
400
500
High Mg
pg/mg protein
Figure 5. Determination of inflammatory response in tissues and plasma. The left sciatic nerve of rats
was injured by crushing. Seven days after injury, (A) the injured nerves (10 mm) were retrieved for the
determination of TNF-␣, IL-1␤, IL-6, and IFN-␥levels. B) Blood was withdrawn for assessment of the
migratory ability of neutrophils and monocytes/macrophages. (C, D) Determination of IL-1␤secretion
in monocytes/macrophages either stimulated with or without LPS.
N=6; *p<0.05; ** p<0.01; *** p<0.001 (relative to basal Mg diet).
study of Mg deficiency in animals, However, after
sciatic nerve injury, many of the mice (about 60-
80%), which were kept in the same cage, were
observed biting other mice. Therefore, the experi-
ments were repeated using a less temperamental
strain of mice (ICR).
Increased nerve regeneration was accompanied
by an improvement in sciatic nerve function index,
increased compound muscle action potential,
reduced nerve conduction latency, and increased
muscle weight [4]. Based on these data, the admin-
istration of high Mg supplementation improved
the neurobehavior of rats, and the low Mg diet
further aggravated the neurological dysfunction.
Axon degeneration took place dramatically 3 to
7 days after nerve crush injury. Evidence indi-
cates that increased expression of neurofilament
reflects early regenerative potential [31]. Nerve
regeneration is also related to Schwann cell proli-
feration in the distal end of nerves as indicated by
increased expression of S-100 [27]. Based on the
early expression of neurofilament and late myeli-
nation marker, treatment with a high Mg diet may
promote greater nerve regeneration, whereas Mg
depletion exacerbates neurological dysfunction.
In addition, increased myelination was positively
correlated to the integrity of nerve tissue and this
reflected the strength of nerve regeneration in
the later phase [22]. In this study, relative den-
sities of NF and S-100 increased 168% and 39%,
respectively, in mice consuming high Mg diets
when compared with those found in the basal
group. However, relative densities of NF and S-100
decreased by 51 % and 36%, respectively, in mice
64

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium promotes nerve regeneration and regulates inflammation
A
B
***
***
*** **
*
*
Basal Mg
Low Mg
14
12
6
4
2
0
0
TUNEL(%)
7 14 Days
8
10
High Mg
Basal
Mg
Low
Mg
High
Mg
Day 3 Day 7 Day 14
Figure 6. Determination of apoptosis levels in crushed nerve. Crushed nerve tissues were retrieved 7
days after injury and were subjected to apoptotic assay by TUNEL.
A) TUNEL positive cells after treatment with three Mg doses at different time points.
B) Quantitative analysis of TUNEL positive cells after treatment with three Mg doses at different time
points.
N=6; *p<0.05; ** p<0.01; *** p<0.001 (relative to basal Mg dose).
Bar length = 50 ␮m. The vertical axis represents the percentage of positive TUNEL assays.
consuming low Mg diets when compared within
the basal group.
An over-activated inflammatory response is a
detrimental stress on the nerve tissues and is a
potential cytotoxic factor in regulation of nerve
regeneration. Macrophages play a central role
in the pathogenic response in peripheral nerves,
and the expression of inflammatory cytokines
65

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
H. C. PAN ET AL.
1Days
AB
bcl-2
bcl-XL
Caspase 3
Cyto C
β tubulin
357 1357 1357
Basal Mg Low Mg High Mg
Basal Mg
Low Mg
High Mg
***
***
***
***
***
*** **
**
4
3.5
3
2.5
2
1.5
1
0.5
0
Intensity arbitary unit
bcl-2
bcl-XL
Caspase-3
Cyto C
Figure 7. Western blot analysis of the retrieved nerve in animals treated with various Mg doses.
A) Illustrative examples of Western blot analysis for bcl-2, bcl-XL, active caspase-3 and cytochrome C
at different time intervals.
B) Graph shows the quantitative results of Western blot analysis with respect to ␤-tubulin.
N=6; ** p<0.01; *** p<0.001 (relative to basal Mg diet).
parallels the number of macrophage deposits [4].
Clearance of macrophage deposits and inhibi-
tion of inflammatory cytokines augmented the
regeneration in peripheral nerve crush injury.
Experimental Mg deficiency is known to have
a profound effect on the process of inflamma-
tion [32, 33]. The underlying mechanisms of
inflammatory response induced by Mg deficiency
have not been clearly elucidated. Several trigger-
ing events highlight possible mechanisms, inclu-
ding (i) cellular entry of calcium and priming
of phagocytic cells; (ii) opening of calcium chan-
nels and activation of NMDA receptors; (iii)
release of neurotransmitters such as substance
P; and (iv) membrane oxidation and activation of
nuclear factor kappa B (NF␬B). Furthermore, Mg
depletion caused the elevation of inflammatory
cytokines in nerve tissue, which was abrogated
by Mg supplements. The alterations of inflamma-
tory cells distributed in nerve tissue paralleled
the alterations in the expression of inflamma-
tory cytokines, and these events were correlated
with the administration of various doses of Mg.
These results were consistent with those of our
previous report that showed Mg modulated the
regulation of Ca++, the priming of inflammatory
cells and possibly the release of inflammatory
cytokines. Monocyte chemoattractant protein 1
(MCP-1) and RANTES are the important regula-
tors of macrophage response that leads to rapid
myelin breakdown and clearance in Wallerian
degeneration. Inhibition of MCP-1 and RANTES
suppresses macrophage deposits and myelin clea-
rance [4]. The expression of MCP-1 and RANTES
was down-regulated in those groups treated with
high-dose Mg supplementation based on one refe-
rence gene (␤-actin). These findings indicate that
a high dose of Mg possesses an anti-inflammatory
effect and depletion of Mg exacerbates the inflam-
matory response in the injured nerve.
The efficacy of stimulation-induced IL-1␤
release was attenuated by a high dose of Mg and
augmented by a low dose of Mg, indicating a
high Mg dosage rendered monocytes/macrophages
insensitive to stimulation of IL-1␤release.
The number of resident peritoneal macrophages
was greater in Mg-deficient rats and these cells
presented the morphological characteristics of
66

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
Magnesium promotes nerve regeneration and regulates inflammation
activated cells. The recruitment of phagocytic
cells and their activity are enhanced in Mg-
deficient animals [17]. In addition, a significant
elevation of pro-inflammatory cytokines, such as
TNF-␣, IL-I, Il-6 and INF-␥, has been reported
in Mg-deficient animals [16-19]. In this study, a
significant increase in deposits of macrophages
on the injured nerve in Mg-deficient rats and
an alleviation of such deposits were found in
animals treated with a high dose of Mg. The
distribution of macrophage deposits was highly
correlated with the expression of macrophage
migration genes such as MCP-1 and RANTES,
and this result was consistent with that of our
previous report [4]. Furthermore, the measure-
ment of pro-inflammatory cytokines retrieved
from the crushed nerve after each treatment also
revealed the same trends. Hence, the intensities of
macrophage deposits and their associated inflam-
matory cytokines were highly correlated with the
concentration of Mg in the nerve tissue. These
results further confirmed the hypothesis that Mg
depletion augments the inflammatory response
and Mg supplementation abrogates it.
Sciatic nerve crush injury produced an imme-
diate spike in macrophage invasion. Macrophage
recruitment was indicated by a statistically sig-
nificant rise in cell numbers as early as 24 hours
after treatment, which reached a peak at 7 days
and declined to normal levels within 21 days [34].
Other studies have reported macrophage recruit-
ment that began at 24 hours after axon injury
and peaked at 14-21 days [35]. The macrophages
in degenerating peripheral nerves are mainly
of haematogenous origin [36, 37]. In this study,
crush injury triggered macrophages to initiate an
increase at 3 days, followed by a plateau at around
7-14 days and a decline to normal values at around
21 days. Mg depletion augmented the macrophage
deposits and Mg supplements alleviated the accu-
mulation of macrophages. The intensity of the
distribution of macrophages in the crushed nerve
was consistent with the macrophage migratory
ability in the blood. Thus, the modulation of
macrophage phagocytic ability by either Mg deple-
tion or supplement significantly contributed to the
distribution of macrophages in the crushed nerve.
The up- or down-regulation of macrophage activity
in the crushed nerve was responsible for the sub-
sequent expression of the inflammatory cytokines.
Macrophages are not only cellular scavengers
phagocytosing remnants of myelin and axon, but
are also very important sources of cytokines.
Macrophages are able to produce the pro-
inflammatory cytokines TNF-␣, IL-1, and IFN-␥
and synthesize neurotrophic factors such as IL-6
and LIF [1, 38, 39]. In this study, upregulation of
the inflammatory cytokines TNF-␣, IL-1, IFN-␥,
and IL6 was demonstrated in animals treated
with a low dose of Mg, and suppression of these
cytokines was observed in animals given a high
dose of Mg. The expression of the inflamma-
tory cytokines was in line with the amount of
macrophage deposits in crushed nerve as well
as the migratory ability of macrophages in the
blood. Taken together, these results demonstrate
that Mg supplementation or depletion primed the
phagocytic ability in the blood and subsequently
altered the deposits of macrophages in crushed
nerve. The intensity of macrophage deposits was
responsible for the expression of inflammatory
cytokines in injured nerve, which in turn deter-
mined the potentiation of nerve regeneration.
Attenuation of Schwann cell apoptosis con-
tributed to significant nerve regeneration. The
ratio of cell apoptosis was highly correlated with
expression of active caspase-3 and cytochrome
C, which was attenuated by high-dose Mg
supplementation and augmented by low-dose
Mg supplements. Taken together, these find-
ings imply that a high dose of Mg exerted an
anti-apoptotic effect on Schwann cells through
significant increases in the expression of bcl-
2 and bcl-XL, which abolished the downstream
expression of active caspase-3 and cytochrome
C. Pro-inflammatory cytokines have been pre-
viously described as potent cytotoxic molecules
during the active phase of demyelination in vari-
ous forms of peripheral neuropathy [3, 4, 10].
TNF-␣generation and secretion are integral pro-
cesses in the series of disease-defining events that
take place during Wallerian degeneration and also
play a central role in regulating the cytokine
network through this process [2]. Furthermore,
administration of TNF-␣to the sciatic nerves
of neonatal axotomized mice has been shown to
increase Schwann cell apoptosis in the distal seg-
ment of injured nerve [12] while depletion of
TNF-␣decreased Schwann cell apoptosis [3, 4].
Apoptosis involves activation of a cascade of pro-
teolytic enzymes called caspases [40]. Members
of the Bcl-2 protein family are key regulators of
apoptosis and are categorized according to their
ability to promote (e.g. Bak, Bax, Bik) or inhibit
(e.g. Bcl-2, Bcl-XL, Bcl-w) apoptosis [41]. Expres-
sion of Bcl-2 and Bcl-XL also inhibits cytochrome
67

Journal Identification = MRH Article Identification = 0280 Date: June 22, 2011 Time: 4:6 pm
H. C. PAN ET AL.
C release but may not interact with pro-caspase-
9 and therefore reduces caspase-3 activation [42].
In this study, Mg depletion increased Schwann cell
apoptosis by decreasing Bcl-2 and Bcl-XL expres-
sion levels and subsequently increasing active
caspase-3 and cytochrome C. Administration of
a high dose of Mg rescued the Schwann cells
from apoptosis by upregulation of the expres-
sion of Bcl-2 and Bcl-XL. However, fractionation
of cytochrome C needs to be clarified either from
the cytosolic or mitochondria cytochrome C. This
further confirmed that Mg supplementation
exerted anti-apoptotic effects against Schwann
cell apoptosis.
Conclusion
Mg depletion induced the release of inflamma-
tory cytokines with a subsequent cascade of
production of macrophage deposits which were
detrimental to nerve regeneration. Mg supple-
mentation suppressed the inflammatory response
and rescued Schwann cells from apoptosis mainly
through upregulation of the anti-apoptotic path-
way of Bcl-2 and Bcl-XL expression. In conclusion,
Mg supplementation may down-regulate inflam-
matory responses and promote sciatic nerve
regeneration.
Acknowledgements
We wish to thank Ms. Shu-Zhen Lai, Ms. Lin-
Lan Yang, and Ms. Mu-Jung Liu for their help
in preparing the manuscript and the Biostatis-
tics Task Force of Taichung Veterans General
Hospital for their assistance with the statistical
analysis.
Disclosure
This study was supported by grants from
Taichung Veterans General Hospital, Provi-
dence University (TCVGH-NCHU987611), and
the National Science Council (NSC-99-2314-B-
075A-005-MY2), Taiwan, R.O.C.
None of the authors has any conflict of interest
to disclose.
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