ORIGINAL ARTICLE

Low doses of 3-nitropropionic acid in vivo induce damagein mouse skeletal muscle

Elizabeth Hernandez-Echeagaray • Nancy Gonzalez •

Angelica Ruelas • Ernesto Mendoza •

Erika Rodrıguez-Martınez • Rafael Antuna-Bizarro

Received: 16 December 2009 / Accepted: 23 July 2010 / Published online: 24 August 2010

� Springer-Verlag 2010

Abstract Mitochondrial alterations are believed to play a

critical role in the pathophysiology of neurodegenerative

diseases and in some well-described myopathies. In the

present study, we evaluated muscle changes in vivo after

blocking the mitochondrial complex II of the respiratory

chain by using 3-nitropropionic acid (3-NP). This neuro-

toxin has been used as a pharmacological tool in animal

models to address some of the metabolic modifications that

might underlie central neurodegeneration; however, chan-

ges in peripheral musculature have not been documented.

We believe that skeletal muscles must be affected because

their integrity highly depends on oxidative metabolism.

Therefore, histochemical, ultrastructural, and biochemical

changes were studied in the muscles of mice treated with

low doses of 3-NP (15 mg/kg, i.p., for 5 days). 3-NP-

treated mice displayed changes in alkaline phosphatase

(APase), succinic dehydrogenase (SDH), and cytochrome c

oxidase (COX) levels in the gracilis and gastrocnemius

muscles. These changes were statistically significant for

APase and SDH in both muscles and for COX only in the

gastrocnemius. No significant alterations in acetylcholin-

esterase (AChE) expression were observed in either mus-

cle. Analysis of the muscle ultrastructure revealed

mitochondrial atrophy as well as sarcomere and nuclei

disorganization. At the biochemical level, nitric oxide

(NO) and lipid peroxidation (LPO) changed in the muscles

of 3-NP-treated mice, suggesting metabolic alterations due

to oxidative stress. Early damage in the striatal tissue and

behavioral modifications are also documented.

Keywords 3-NP � Muscle � SDH � Neurodegeneration �Oxidative phosphorylation

Introduction

Substantial evidence indicates that one of the underlying

mechanisms of neurodegeneration is a defect in bioener-

getics; in particular, failures in mitochondrial complexes II,

III, and IV may play a role in the pathogenesis of neuro-

degenerative disorders in humans and in animal models

of neurodegeneration [1–4]. The mitochondrial toxin

3-nitropropionic acid (3-NP) has been widely used to

unravel the degenerative process underlying Huntington’s

disease (HD) because its damage replicates with high fidelity

the neuropathological hallmarks seen in patients afflicted

with HD [5]. 3-NP blocks succinic dehydrogenase (SDH)

in mitochondrial complex II and produces pathological

changes in striatal tissue. Thus, 3-NP is a pharmacological

tool that can reproduce the phenotype of disorders that

exhibit striatal degeneration due to a mitochondrial dys-

function, such as HD, type I glutaric aciduria, and certain

inherited metabolic disorders [6]. Nevertheless, a defi-

ciency in energy metabolism may damage non-neuronal

tissue as well. For example, muscles need the energy

supply from glycolysis and oxidative phosphorylation to

participate in locomotion, heat production, and overall

metabolism [7]. Indeed, neurodegenerative diseases that

E. Hernandez-Echeagaray (&) � N. Gonzalez � A. Ruelas �E. Mendoza � E. Rodrıguez-Martınez

Laboratorio de Neurofisiologıa del Desarrollo y la

Neurodegeneracion, Unidad de Biomedicina, FES-I,

Universidad Nacional Autonoma de Mexico,

Av. De Los Barrios # 1, Los Reyes Iztacala,

C. P. 54090 Tlalnepantla, Mexico

e-mail: elihernandez@campus.iztacala.unam.mx;

ehe67@yahoo.com

R. Antuna-Bizarro

Instituto Nacional de Pediatrıa, C. P. 04530 Mexico city, Mexico

123

Neurol Sci (2011) 32:241–254

DOI 10.1007/s10072-010-0394-2

affect the nervous system exhibit changes in other parts of

the organism [8]. In particular, humans who develop HD

show many sensory and motor deficits [9–11], with weight

loss being a characteristic feature [12]. In the transgenic

mouse model of HD, protein inclusions [13] and mutant

huntingtin protein are found in skeletal muscle [14]. Motor

impairments may directly result from central neurodegene-

ration; however, it is also possible that peripheral distur-

bances could reflect a more general metabolic failure. If

true, this reasoning could explain, in part, the muscle

atrophy and body weight loss documented in HD patients

[12, 15], in transgenic HD mice [13, 14] and in patients

with metabolic dysfunction disorders [16, 17].

Until now, most of the studies carried out using the

mitochondrial neurotoxin 3-NP focused on evaluating

central damage [5, 6, 18, 19] even though in a number of

studies 3-NP was systemically administered [20]. A pio-

neering study on 3-NP toxicity in non-neural tissue

revealed a cardiac pathology in mice associated with

changes in succinate dehydrogenase activity [21]. Because

a breakdown in mitochondrial function induces oxidative

damage in mouse muscle [22], in this study, we wanted to

address the potential changes that 3-NP might generate

outside the brain. We believe that documenting modifica-

tions in skeletal muscle after uncoupling oxidative

metabolism provides insights about how early metabolic

failures may produce modifications in non-neural tissue,

which in turn could explain body weight changes observed

in neurodegenerative diseases. Therefore, in the present

study, histochemical, biochemical, and morphological

changes in mouse skeletal muscles were evaluated after

chronic inhibition in vivo of mitochondrial complex II with

low doses (15 mg/kg, i.p., for 5 days) of 3-NP in C57BL/6

mice. Besides the peripheral evaluations, striatal tissue was

processed for a TUNEL assay to document cellular damage

in the brain, and behavioral evaluations were carried out to

address motor disturbances.

Materials and methods

Animals

This study employed 30 juvenile C57BL/6 male mice

(30-days-old) bred by Harlan (Mexico) and housed at the

UBIMED, FES-I, UNAM, throughout the 3-NP treatment

period. The animals were kept on a 12-h/12-h light/dark

cycle with free access to food and water. Body weight was

measured during the 3-NP treatment period. The experi-

mental protocol followed the regulations established by the

Institutional Animal Care of the National Autonomous

University of Mexico and the NIH Guide for the Care and

Use of Laboratory Animals.

3-NP treatment

Fifteen mice were treated for 5 days with 3-NP (15 mg/kg,

i.p., 7.4 pH), and 15 control group animals were treated with

drug vehicle (phosphate buffer, 7.4 pH). On the sixth day,

animals were neurologically and behaviorally evaluated. On

the seventh day, 2 days after the last drug treatment, changes

in the hind limb muscle innervations, vascular supply, and

mitochondrial enzyme activity were evaluated with histo-

chemical techniques. Ultrastructural changes at the muscle

were documented with electron microscopy (EM), and oxi-

dative stress was addressed with biochemical procedures.

Muscle dissection

The mice used for histochemical evaluations (n = 10; 5 for

3-NP and 5 for control) were killed by cervical dislocation

under deep anesthesia induced by ether inhalation. Gas-

trocnemius and gracilis muscles were quickly excised from

both hind limbs and dissected from the deep part of the

lateral head and from the superficial part of both lateral and

medial heads. Muscle samples were immediately immersed

in cooled methylbutane liquid and stored at -80�C for

subsequent analyses.

Histochemical assays

Histochemical procedures were carried out in ten mice

(five muscles from 3-NP and five muscles from control; the

remnant muscles were used for immunohistochemical

evaluation). Fresh, frozen, transverse sections (20 lm)

taken throughout the midbelly region of the muscle were

obtained with a cryostat. Tissue sections were mounted on

different glass slides depending on the histochemical

analysis (SDH, COX, APase or AChE). The distance

between tissue sections on a glass slide for any histo-

chemical assay was 60 lm.

Succinate dehydrogenase (SDH)

SDH staining was used to identify slow contraction (oxi-

dative phosphorylation) from fast contraction (glycolytic)

fibers. Muscle slices used for SDH were incubated in

polyvinyl-alcohol, phosphate buffer (PB, 0.1 M, pH 7.4),

sodium succinate, and sodium azide in the presence of nitro

blue tetrazolium and phenazine methosulfate for 15 min on

a light-protected, cold surface. The slices were then rinsed

with distilled water.

Cytochrome c oxidase (COX)

COX staining also identifies oxidative phosphorylation

activity. Tissue slices used for COX were incubated for

242 Neurol Sci (2011) 32:241–254

123

12–15 h in PB (0.1 M, pH 7.4) containing 130 mM

sucrose, 6.702 lM cytochrome c, 100 lg/ml of catalase

units from bovine liver, and 1.54 mM 3,3-diaminobenzi-

dine at 37�C. After the incubation period, the slices were

rinsed three times in distilled water.

Alkaline phosphatase

Alkaline phosphatase (APase) was used to identify vascular

endothelium and evaluate changes in the muscle’s vessel

supply. The muscle slices used for APase were incubated in

the Blue Alkaline Phosphatase Substrate Kit III SK-5300

for 25 min. Afterwards, the slices were transferred into a

freshly prepared Tris–HCl buffer (100 mM, pH 8.5) for

5 min and later rinsed with distilled water.

Acetylcholinesterase

Motor endplates were visualized with the Acetylcholin-

esterase (AChE) staining. The muscle slices used for

AChE were incubated overnight in the dark at room

temperature in sodium acetate buffer (50 mM, pH 5.0,

adjusted with HCl) containing sodium acetate (0.1 M),

cupric sulfate (30 mM), glycine, acetylthiocholine iodide

(5 mg), and ethopropazine. The following day, the slices

were rinsed with distilled water and treated with 10%

solution of Na2S H20, pH 7.5 (adjusted with acetic acid),

for 5 min and later rinsed with distilled water. All slides

for histochemical assays were rinsed with distilled water

after the enzymatic reaction, dried, and covered with

Cytoseal TM 60.

Immunohistochemical identification of fiber type

Expression levels of myosin heavy chain (MHC) subtypes

were documented using Immunohistochemical (IHC) pro-

cedures. Remnant muscles from histochemical evaluations

(five from control and five from 3-NP-treated mice) were

used. Cryostat transverse sections (10 lm) of frozen mus-

cles were cut from the midbelly region of each muscle and

immunostained using monoclonal antibodies specific for

MHC. Briefly, sections were fixed in 4% paraformaldehyde

in PB (pH 7.4) for 2 h and then incubated at 4�C in

blocking serum (3% bovine serum albumin, 0.3% triton

X-100 and 0.025% sodium azide in phosphate buffer, pH 7.4)

for 12 h before being incubated overnight at 4�C with slow

type (myosin type 1, A4.951, 1:100) and fast type (myosin

type 2, N3.36, 1:100 and myosin type 2A, A4.74, 1:100,

Santa Cruz Biotechnology, Inc.) MHC antibodies in

blocking solution. Then, sections were washed three times

with phosphate-buffered saline (PBS). Biotinylated sec-

ondary antibody (1:500, Vector Laboratories, Inc., Bur-

lingame, CA, USA) in blocking serum was added for

90 min at room temperature. Sections were rinsed with

PBS, and the AB Vector kit (Vector Laboratories, Inc.,

Burlingame, CA, USA) was used to detect biotinylated

antibodies following the manufacturer’s instructions. Tis-

sue slices were cover slipped with mounting medium

(Fisher Scientific, Pittsburgh, PA, USA). Control experi-

ments were carried out by pre-absorbing the primary

antibodies with their corresponding antigens, omitting the

primary antibody or omitting both the primary and sec-

ondary antibodies. Counting of fiber types was performed

over the entire muscle cross-sections to establish the

number of each fiber type in relation to the total fiber

number. A total of six muscles collected from three control

mice and three mice treated with 3-NP were analyzed.

Electron microscopy

Animals were perfused with paraformaldehyde (4%) plus

glutaraldehyde (2.5%) and later postfixed with glutaralde-

hyde (2.5%) in a cacodylate buffer (pH 7.0, 0.1 M) for

1.5 h. Muscles were excised as previously described and

then rinsed for 10 min and postfixed with osmium tetroxide

(OsO4, 0.5%) in cacodylate buffer. OsO4 was rinsed with

cacodylate buffer alone, and the tissue was dehydrated by

immersion in increasing alcohol concentrations (70�, 80�,

90� and 100�) and propylene oxide. Tissue was pre-

embedded in epon-propylene oxide, 1:1, overnight and

later embedded in pure epon�. Tissue slabs of 1 lm were

stained with toluidine blue. Subsequently, ultra-thin

(60–70 nm) slices were contrasted using uranyl acetate

(3%) and lead citrate for visualization. Electron micro-

graphs were taken for further analysis. Brains from these

perfused mice were used for the TUNEL assay to evaluate

apoptotic death.

Evaluation of oxidative stress induction

NO

Nitric oxide production was evaluated indirectly by mea-

suring nitrates in the serum from blood samples. Blood

samples from 3-NP and control mice were centrifuged at

12,000 rpm for 15 min to separate nitrates from the serum.

To quantify nitrates, 0.1% of naphthyl ethylenediamine

dihydrochloride was mixed with Griess reagent (1% sul-

fanilamide and 5% phosphoric acid). In a plate of 96 wells,

serum samples (100 ll, different concentrations) and 75 ll

of Griess reagent were incubated at room temperature for

10 min and measured at 550–620 nm in an ELISA reader.

Absorbance spectrums of the colored azo compounds were

quantified using serial dilutions of sodium nitrite as the

standard reference curve. Data are expressed as absorbance

units.

Neurol Sci (2011) 32:241–254 243

123

LPO

Lipid peroxidation was measured using the lipid fluores-

cent products. Briefly, muscle from control (n = 3) and

3-NP-treated (n = 3) mice was weighed and homogenized

in 3 ml of saline solution (0.9% NaCl). Aliquots of 1 ml

were added to 4 ml of a chloroform–methanol mixture (2:1

v/v). After stirring, the mixtures were kept on ice for

30 min to permit phase separation, and then the fluorescent

of the chloroformed layer was measured in a Perkin–Elmer

LS50B luminescence spectrometer at 370 nm of excitation

and 430 nm of emission wavelength. The sensitivity of the

spectrometer was previously adjusted with a quinine stan-

dard solution (0.1 lg/ml). Results are expressed as fluo-

rescent units/g of wet tissue.

Evaluation of striatal damage

TUNEL

The brains of six perfused mice were dissected and

cryostat-cut in coronal sections (5 lm) to detect DNA

fragmentation with the TUNEL reaction kit (Apoptosis

Detection Kit, TA300, R&D Systems, Minneapolis, MN,

USA). In brief, slices were mounted on glass slides and

postfixed with formalin (10%) in 0.1 mM PBS and later

incubated in proteinase K (20 lg/ml) in 10 mM Tris–

HCl, pH 7.5, for 15 min at 18–24�C. After rinsing the

sections in distillated water, quenching solution (methanol

in 30% H2O2) was added for 5 min at 18–24�C and then

washed with distilled water and equilibrated for 5 min in

terminal deoxynucleotidyl transferase buffer (50 mM

Tris–HCl, pH 8.0, 1 mM MgCl2, 0.5 mg/ml BSA,

0.6 mM 2-mercaptoethanol sulfonic acid) at 18–24�C.

The labeling reaction was performed at 37�C for 1 h with

the Labeling Reaction Mix from the detection kit. The

reaction was stopped by using the TdT Stop Buffer for

5 min at 18–24�C. The tissue was washed twice for

15 min in distillated water, incubated with 50 ll of

diluted anti-BrdU for 1 h at 37�C, and then washed three

times with PBS-Tween 20 (0.05%). TUNEL labeling was

carried out using 50 ll of Streptavidin-HRP Solution for

10 min at 18–24�C. Samples were rinsed with distilled

water to stop the reaction and covered with 50 ll of

TACS Blue Label for 2–5 min. Finally, the tissue was

rinsed, dried, and cover slipped with Permount (Fisher

Scientific, Pittsburgh, PA, USA). TUNEL-labeled cells

that displayed densely labeled, small particles in the

cytoplasm (apoptotic bodies) and different types of

chromatin condensation around the margin of the nucleus,

forming either crescent caps or rings, were quantified by

light microscopy.

Cell counting

To count damaged cells stained by the TUNEL labeling,

images were visualized and captured with a microscope

equipped with a video camera coupled to a computer

monitor, and then the digital images were analyzed (Image

J, NIH). The analysis consisted of obtaining square probes

of 1 mm2 in 409 magnification to determine labeled cells.

The number of cells was obtained as an average of three

fields per section, ten sections per brain, and three mice per

experimental group.

Evaluation of motor disturbances

Motor disturbances were classified in terms of the presence

of dystonia and gait abnormalities. Dystonia classifications

consisted of 0 = none, 1 = intermittent dystonia of one

limb, 2 = intermittent dystonia of two limbs or permanent

dystonia of one limb, and 3 = permanent dystonia. Clas-

sifications for gait abnormalities were 0 = none and

1 = uncoordinated and wobbling gait.

Behavioral evaluations

We also evaluated orofacial dyskinesia and measured the

latency to paw clasping. To evaluate orofacial dyskinesias

in mice, spontaneous mouth movements were scored for

10 min by a hand-operated counter. Mice were observed

individually in their own Plexiglas cages, and mirrors were

placed at the back wall of the cage to permit the assessment

of vacuous chewing movements when mice were facing

away from the observer. To the latency to paw clasping,

mice were suspended by their tail above 10 cm from their

cages up to 120 s. Results are expressed as the mean ±

standard error.

Data analysis

Fiber number was counted in transverse muscle cross sec-

tions, taken at the middle of the muscle (five muscles for

each group), and then images of 2,000 lm2 per field were

analyzed (3–5 microscopic fields for each muscle). For

optical density analysis, the images of muscles were all

captured maintaining the same values for brightness and

gain. Ten sections per animal were captured and digitized to

estimate histochemical reactions for COX, SDH, APase and

AChE in control, and 3-NP-treated mice. Optical density

analysis was performed through a computer-assisted

imaging analysis system (Scion Image; Scion Corp, Beta

4.0.2). The optical density was measured only in transverse

sections of the gastrocnemius muscle to analyze the

differential reactivity of red and white fibers for SDH, and

244 Neurol Sci (2011) 32:241–254

123

COX histochemical activity; for AChE and APase expres-

sion, we evaluated both gastrocnemius and gracilis muscles.

The muscle data obtained from each mouse were averaged,

and statistical comparisons were made between experi-

mental and control groups using an unpaired Student’s t test

for two-group comparisons or a Mann–Whitney Rank Sum

Test for data where normality tests did not pass. All data

except that documenting striatal damage induced by 3-NP

were expressed as mean ± S.E.M. Statistical analysis was

conducted using SigmaStat statistical software for Win-

dows, version 2.03 (Systat Software, Inc.), and p \ 0.05

was considered to be statistically significant.

Reagents

3-Nitropropionic acid, sodium acetate, cupric sulfate, gly-

cine, acetylthiocholine, ethopropazine, sodium sulfite,

cytochrome c, catalase from bovine liver, 3,3-dıamino-

benzidine, polyvinyl-alcohol, sodium succinate, sodium

azide, nitroblue tetrazolium, phenazine methosulfate, and

paraformaldehyde were purchased from Sigma Chemical

Co. (St. Louis, MO, USA). Glutaraldehyde, osmium

tetroxide, propylene oxide and uranyl acetate were pur-

chased from Merck Co., Inc. (Whitehouse Station, NJ,

USA). Toluidine blue was from EMS (Electron Micro-

scopic Sciences, Hatfield, PA, USA). Blue alkaline phos-

phatase substrate Kit III SK-5300 was from Vector

(Vector�, SK-5300). Tris–HCl was from BioRad (Hercu-

les, CA, USA). Sucrose was from JT Baker, Inc. (Mal-

linckrodt Baker, Phillipsburg, NJ, USA), and Cytoseal

TM60 was from Richard-Allen Scientific (Kalamazoo, MI,

USA).

Results

In our experimental conditions, we did not observe body

weight changes (F6.629, p [ 0.062, one-way repeated

measures analysis of variance) or motor disturbances in

animals treated with 3-NP when compared with the control

group; however, signs of muscle alterations were docu-

mented through histochemical analysis, electron micro-

graphs and biochemical evaluations. Striatal damage

and changes in some behavioral parameters were also

documented.

Histochemical evaluations

Succinate dehydrogenase

We found small-diameter muscle fibers with reddish color

that were positive for SDH staining and large diameter

fibers with light color that were negative for SDH staining.

We evaluated the SDH-positive, small size fibers and found

a significant increase in the number of fibers stained for

SDH in gastrocnemius (t45 = -3.845, p = 0.001) and

gracilis (t49 = -5.585; p = 0.001) from 3-NP-treated

animals when compared with controls (Figs. 1a, b, 2a).

Optical density measurements of the SDH staining

showed greater intensity in the small fibers (t22 = -3.214,

Fig. 1 SDH and COX-positive

fibers increased in muscle of

3-NP-treated animals. a shows

SDH histochemical staining in

transverse sections of the

gastrocnemius muscle in the

control condition. SDH stains

small fibers (white arrow),

typical for slow contraction

fibers. Optical density and the

number of SDH-positive fibers

increased in 3-NP-treated

animals (b). c shows the COX-

positive fibers in control

gastrocnemius muscle, whereas

d displays the muscle of 3-NP-

treated animals. COX staining

increases in 3-NP-treated mice,

and it was present in both the

white and the red areas of the

muscle. Arrows show the red

area in both cases. Scale bar20 lm

Neurol Sci (2011) 32:241–254 245

123

p = 0.004) of animals treated with 3-NP than in their

respective controls (Table 1). We did not observe statistical

changes in the diameter size of SDH positive fibers

between the groups; however, the size of the white

fibers (SDH-negative fibers) was increased significantly

in 3-NP-treated animals (24.15 ± 1.83 lm) compared

with the control group (19.73 ± 1.02 lm) (t54 = -2.283,

p = 0.026).

Cytochrome c oxidase

The number of COX positive fibers increased significantly

(t51 = 2.437, p = 0.018) in the gastrocnemius muscle of

3-NP-treated mice without a significant change in the gracilis

muscle (Figs. 1c, d, 2b). Optical density analysis for COX

positive fibers in the gastrocnemius muscle also showed

statistical differences (t269 = -3.046, p = 0.003) in 3-NP-

treated animals vs. controls (Table 1). Note that COX

staining fibers increased in both the white and red areas of

the 3-NP-treated muscle (Fig. 1d).

Alkaline phosphatase

APase histochemical expression was used to evaluate the

vascular supply in the muscle. The number of stained

capillaries significantly increased in 3-NP animals in both

the gastrocnemius (t29 = -2.351, p = 0.026) and gracilis

muscles (t67 = -2.254, p = 0.027) when compared with

controls (Figs. 2c, 3a, b).

Acetylcholinesterase

We used the Tago’s method to visualize motor end plates.

The histochemical expression of AChE did not differ sig-

nificantly in either the gastrocnemius or the gracilis mus-

cles between control and treated animals (Figs. 2d, 3c, d).

Muscle atrophy

Structural and biochemical evaluations were centered only

in the gastrocnemius muscle of the control and 3-NP-treated

mice because there were no differences between the gracilis

and gastrocnemius muscles in the histochemical evaluations.

We chose to analyze the muscle ultrastructure with the aid of

EM. The micrograph of the control muscle displays a normal

appearance in its nuclei and sarcomere structure (Fig. 4a, c),

but 3-NP-treated muscles displayed changes in their cell

nuclei, which showed early signs of apoptotic damage when

compared with control muscle (Fig. 4b, white arrow). 3-NP-

treated muscles exhibited a disorganization of the sarco-

mere; in particular, Z bands were remarkably thicker

Fig. 2 Summary of SDH,

COX, APase, and AChE

histochemical expression in

mouse muscles. a shows SDH

positive fibers. b shows COX

positive fibers. c shows APase

expression in capillaries, and

d shows AChE expression in the

endplates of the muscle. a and

b shows that 3-NP-treated mice

increased their mitochondrial

enzymatic activity in both

gastrocnemius and gracilis

muscles. Capillary number

visualized with APase

histochemistry also increased in

3-NP-treated animals in both

gastrocnemius and gracilis

muscles (c). Endplate number in

gastrocnemius and gracilis

muscles did not display

differences between control and

3-NP-treated mice

(d) (*p \ 0.05)

Table 1 Optical density measurements

Group SDH (pixels) COX (pixels)

Control 100 ± 7.98% 100 ± 3.32%

3NP 141.81 ± 9.02%* 112.30 ± 2.44%*

* p B 0.005 Data are expressed as percentages from control ± SEM

246 Neurol Sci (2011) 32:241–254

123

(Fig. 4d, white arrow) than in control muscle (Fig. 4c, white

arrow). Also, M bands were weak in treated animals (Fig. 4d,

black arrow) compared with controls (Fig. 4c, black arrow).

In fact, some M bands were difficult to visualize in the

muscle of 3-NP-treated mice in contrast to the thickness of

their Z band. In addition, in the muscles of 3-NP-treated

mice, there was a notable augmentation in the number of

mitochondria measured per field in 7,0009. We evaluated

the SDH-positive micrographs (control 20.66 ± 6.16 vs.

138.16 ± 24.30 for 3-NP-treated mice, t10 = 4.687,

p \ 0.001), and their morphology showed signs of atrophy

(Fig. 4d, dashed black arrow; see also Fig. 5b, d) when

compared with the mitochondria in control muscles (Fig. 4c,

dashed black arrow; Fig. 5a). Figure 5 shows changes in the

amount of mitochondria in muscle from control and 3-NP-

treated mice. Mitochondria were densely distributed near the

cell nuclei in muscles from 3-NP-treated animals (Fig. 5b,

d), but not in control muscle (Fig. 5a, c). At higher magni-

fication, mitochondrial fission (Fig. 5d, white arrows),

atrophied mitochondria (Fig. 5d, black arrow), and vacuoles

(Fig. 5d, dashed black arrow) could be visualized. Finally,

Fig. 5b exhibits a dividing sarcomere (white arrow).

Fiber type examination

To further analyze if changes observed in muscle tissue were

due to the effects of 3-NP treatment on fiber composition,

muscle cryosections were immunostained with specific

myosin antibodies. The percentage of slow contraction fibers

(Type I) did not change in 3-NP-treated mice in comparison

with control mice (t18 = 1.857, p = 0.080). Fast contraction

fibers did not change in 3-NP-treated mice with respect to

control mice (Type II fibers, t18 = -0.360, p = 0.723; Type

IIA fibers, t18 = 0.866, p = 0.398, see Table 2).

Biochemical evaluations

Nitrates and Nitrite levels in the muscle of 3-NP-treated

animals

The inhibition of oxidative phosphorylation may trigger an

increase in reactive oxygen species (ROS), inducing oxi-

dative stress and neuronal damage. To investigate if oxi-

dative stress underlies the 3-NP effects on muscle

alterations, we evaluated the presence of NO in the muscle

indirectly by measuring NO3-/NO2

- production. Levels

of NO3-/NO2

- increased significantly (t12 = -3.846,

p = 0.002, Fig. 6a) in 3-NP-treated mice (0.125 ± 0.009,

absorbance units) in comparison with control group

(0.07 ± 0.007, absorbance units).

LPO in 3-NP-treated animals

One of the harmful effects of the increase in NO levels is

the oxidation of fatty acids, a process called lipid peroxi-

dation. Therefore, LPO levels were also estimated. Our

evaluation demonstrated a significant increase in the LPO

levels in the gastrocnemius muscle of 3-NP-treated mice

Fig. 3 APase and AChE

histochemical staining. a shows

the alkaline phosphatase

histochemical reaction to

visualize capillaries in a

transverse section of the

gastrocnemius muscle in control

conditions. b exhibits muscle

from 3-NP-treated animals

(arrows point to capillaries).

c shows the histochemical

expression of AChE in

transverse sections of the

gastrocnemius muscle in the

control group to visualize

endplates, and d exhibits AChE

expression in the 3-NP-treated

animals. Scale bar 20 lm

Neurol Sci (2011) 32:241–254 247

123

(3-NP, 9.87 ± 0.63 vs. control 6.32 ± 0.39, t10 = 4.725,

p \ 0.001, Fig. 6b).

Central damage

Apoptotic damage in the striatum of 3-NP-treated animals

To evaluate if the same low doses of 3-NP that induced

muscle alterations also triggered apoptotic damage in the

striatum, a TUNEL assay was carried out. Figure 7a

illustrates representative images obtained from brain slices

of both control and 3-NP-treated mice. Figure 7b shows

that 3-NP significantly increased the TUNEL positive cells

in comparison with the control tissue (Mann–Whitney,

T = 5,945.500, p \ 0.004).

Behavioral assessment

Neurological evaluation of motor disturbances, such as gait

and dystonia, in the 3-NP-treated animals did not display

differences compared with the control mice. Alternatively,

we chose to study the presence of stereotyped and unco-

ordinated movements through the evaluation of orofacial

dyskinesia incidence and the latency for clasping behavior.

Orofacial dyskinesia

Figure 8a shows that, in a period of 10 min, 3-NP-treated

mice exhibited a significant increase (t18 = -9.287,

p \ 0.001) in orofacial dyskinesias (10.7 ± 0.3) over

control mice (6.3 ± 0.3).

Clasping behavior

Mice from the control and 3-NP-treated groups were also

evaluated on a tail suspension test. Control mice exhibited

a higher latency to clasp (150.4 ± 5.5 s) than 3-NP-treated

mice (79.1 ± 4.3 s), which was significant (t18 = 10.104,

p \ 0.001) (Fig. 8b).

Fig. 4 Ultrastructural analysis

of muscle. a (44009) and

c (20,0009) display electron

microscopy of the

gastrocnemius muscle from

control and b (4,4009) and

d (20,0009) show

gastrocnemius muscle from

3-NP-treated animals. The whitearrows in a and b indicate

changes at the cell nuclei. Note

the membrane invaginations in

the cell nuclei in b. c shows

normal sarcomere organization.

The M band is pointed out with

a black arrow; note that the M

band almost disappears in some

sarcomeres of 3-NP-treated

mice (d, black arrow), but the Z

bands get thicker (d, whitearrow) in the 3-NP-treated mice

in contrast to the control mice

(c, white arrow). The dashedarrow in d shows changes in the

mitochondrial number and

morphology. Scale bar 500 nm

248 Neurol Sci (2011) 32:241–254

123

Discussion

The present study demonstrated that juvenile mice treated

chronically with low concentrations of 3-NP have altered

muscle enzymatic staining for SDH, COX, and vascular

supply without changes in acetylcholinesterase activity in

both gastrocnemius and gracilis muscles. In addition, an

analysis of muscle ultrastructure revealed sarcomere dis-

organization, increased mitochondria number, and mor-

phological modifications in the gastrocnemius muscle of

3-NP-treated animals. The underlying mechanism of these

muscle changes might be due to the generation of oxidative

stress because 3-NP increased NO and LPO levels in the

muscle.

Mitochondrial enzyme activities

To monitor the activity of oxidative enzymes in our

experimental procedures, we evaluated SDH and COX

with histochemical procedures.

Succinic dehydrogenase

SDH is an enzyme of the respiratory chain and the tricar-

boxylic acid cycle [23]; it is used to identify oxidative

muscle fiber subtypes by its reddish staining [24]. As

previously described, we found a strong staining in small

muscle fibers and weak or no staining in fibers with large

Fig. 5 3-NP increases

mitochondrial number in the

gastrocnemius muscle.

a (7,0009) and c (12,0009) are

micrographs of control muscle.

There is no mitochondrial

proliferation near the cell nuclei

(a, black arrow). b (7,0009)

and d (12,0009) are

micrographs of gastrocnemius

muscle of 3-NP-treated mice.

b shows massive increase of

mitochondria, mainly located

near the cell nuclei. Note that

the Z bands of the sarcomere are

thick. The white arrow in

b shows an area where the

sarcomere is splitting. c and

d are magnifications of the

squares in a and b. In d, the

white arrows show

mitochondrial fissions, and the

black dashed arrow shows a

vacuole in a mitochondria.

Scale bar 500 nm

Table 2 Fiber type

Fibre type Control 3-NP

Type I 14.5 ± 0.2% 13.9 ± 0.2% t18 = 1.857, p = 0.080

Type II 76.9 ± 0.4% 77.1 ± 0.2% t18 = -0.360, p = 0.723

Type IIA 9.6 ± 0.1% 9.4 ± 0.1% t18 = 0.866, p = 0.398

Data are expressed as Mean ± SEM

Neurol Sci (2011) 32:241–254 249

123

diameter when evaluated with the SDH histochemical

assay [24]. Optical density analysis of SDH-positive fibers

in the muscle of 3-NP-treated animals showed an increase

in its activity and an increase in the number of SDH-

positive fibers in both gastrocnemius and gracilis muscles

in comparison to control mice. In contrast, the SDH-nega-

tive fibers displayed an increase in diameter; this fact is

important in regard to muscle physiology. It is known that

red fibers (high hemoglobin content), also known as Type I

fibers, show slow contraction [25], elevated oxygen

Fig. 6 3-NP induces oxidative

stress in the gastrocnemius

muscle. a shows that nitric

oxide levels measured indirectly

by nitrite production increased

in the 3-NP-treated muscle.

b shows that lipid peroxidation

also increased in the

3-NP-treated muscle in

comparison with control

muscle (**p \ 0.005)

Fig. 7 Low doses of 3-NP

induce cell damage in mouse

striatal tissue. The striatal tissue

from mice treated with low

doses of 3-NP exhibited

significantly more apoptotic

cells than the control group,

estimated using the TUNEL

assay (**p \ 0.005)

250 Neurol Sci (2011) 32:241–254

123

demands, and use oxidative metabolism as an energy

resource. Therefore, these fibers have high amounts of

mitochondria [26]. After blocking endogenous SDH with

3-NP, it is likely that the first mechanistic change in the

muscle was to produce more endogenous SDH to coun-

teract the SDH inhibition by 3-NP. It is known that mito-

chondrial complex II subunits are replaced in about 12 h

[27]. As we evaluated animal tissue 48 h after the last

doses of treatment, the system had time to synthesize new

SDH.

To evaluate if the increase in the diameter size of SDH-

negative fibers was associated with a transition in muscle

fiber composition due to changes in neuronal innervation

[7] or metabolic activity [28], immunocytochemical anal-

yses of myosin subtype were conducted, revealing that

fiber type composition was similar in muscles from control

and 3-NP-treated mice. These results are comparable to

what has been reported in the transgenic mouse model of

HD [13]. Alternatively, glycolytic activity may have

changed in the white fibers; we have seen an increase in the

mRNA of the glycolytic enzyme GADPH after 3-NP

administration in both striatal and muscle tissues (unpub-

lished data). White fibers (Type II fibers) are fast con-

tracting, have low levels of hemoglobin, have few

mitochondria, and need fast energy sources derived from

glycolysis [25]. Because oxidative phosphorylation was

blocked, it is likely that glycolysis was favored. This

explanation is consistent with the idea that muscle fibers

can change their energy source depending upon energy

demands without changing fiber type composition [28].

Further experiments need to be done to address this point.

Cytochrome c oxidase

COX is an oxidative enzyme, and its activity has been used

as a marker of oxidative metabolism in the brain [29]. Also,

in animals where mitochondrial biogenesis has been doc-

umented, COX expression is increased [30]. Therefore, we

evaluated its histochemical expression and found a significant

increase in COX activity in the muscle of 3-NP-treated

mice, indicating an increase in both mitochondrial activity

and oxidative phosphorylation (Fig. 1).

Changes in vascular supply

To evaluate the vascular supply, we used the alkaline

phosphatase histochemical assay, which stains endothelial

cells in capillaries [31] and has been used to evaluate cap-

illary supply [32]. The amount of stained capillaries

increased in 3-NP-treated animals in both gastrocnemius

and gracilis muscles. This increment can be due to an

increase in vasculogenesis produced by the hypoxia derived

from the inhibition of complex II in the mitochondria.

During vessel development, a mechanism based on hypoxia

is responsible for the release of angiogenetic growth factors

producing angiogenesis [33]; therefore, it is possible that in

adult muscle vasculogenesis is triggered during hypoxia,

oxygen deprivation, or inhibition of oxidative phosphory-

lation because the microcirculatory system is important for

providing optimal metabolic conditions and oxygen supply

to the muscle cells [26, 34]. The lateral gastrocnemius

muscle has a glycolytic fiber angiotype, but its medial part

has a mixture of oxidative and glycolytic fibers [35, 36].

Consequently, if oxidative phosphorylation was blocked, it

is likely that vasculogenesis led to an increase in the number

of capillaries to enhance oxygen supply.

Compensatory mechanisms after oxygen deprivation

In the present study, we observed a link between mito-

chondrial complex II inhibition and the enhancement of

SDH, COX, and alkaline phosphatase histochemical stain-

ing in 3-NP-treated animals, suggesting that more blood

supply was needed as a result of the oxidative phosphory-

lation blockage. We speculate that, after oxidative phos-

phorylation inhibition, the organism elevates its enzymatic

activity related to oxidative phosphorylation to counteract

any metabolic failure. Some studies report the initiation of a

protective mechanism after a brief inhibition of SDH with

3-NP, and this condition seems to depend on the timing of

Fig. 8 Low doses of 3-NP

induced changes in the numbers

of orofacial dyskinesias and

delayed the manifestation of

clasping behavior. Orofacial

dyskinesias (a) increased in 3-

NP-treated mice, and the

latency to clasp (b) was reduced

in comparison with the control

group. Changes were significant

in both cases (**p \ 0.005)

Neurol Sci (2011) 32:241–254 251

123

metabolic perturbations. For example, a short ischemic or

hypoxic episode may increase or decrease tolerance to a

subsequent cerebral or cardiac ischemic event. This process

is called chemical preconditioning. While a short-time

interval of preconditioning is needed to induce heart toler-

ance [21], in the brain, it may last a few days after a single

dose of 3-NP. In our study, 3-NP was administered for five

consecutive days in low doses, but further manipulations

such as ischemic manipulation were not carried out. Thus,

we doubt that chemical preconditioning is responsible for

our data. Also, it is known that complex II inhibition is not

necessary for chemical preconditioning [37]. It is also pos-

sible that the changes observed occurred because C57BL/6

mice are less susceptible to this mitochondrial toxin. If this is

true, the use of higher doses could result in a reduction of

enzymatic activity as previously demonstrated in cardiac

muscle [21]. However, we were not interested in analyzing

cellular processes in the brain and in non-neuronal tissue

when massive damage was produced. Instead, we studied the

early changes in neurodegeneration to stop or delay neuronal

and non neuronal damage, and consequently, we needed

lower doses of 3-NP in our studies. We support the idea that,

after inhibiting oxidative phosphorylation, the first mecha-

nism the body develops is the increase in mitochondrial

activity and mitochondrial number to counteract mito-

chondrial failure.

Mitochondrial increase

Histochemical assays did not give direct information about

the presence of mitochondrial damage after blocking com-

plex II of the respiratory chain, but EM visualization showed

both mitochondrial atrophy and an increase in mitochondrial

number. The increase in mitochondrial number correlates

very well with the SDH staining increase. Both the mito-

chondrial atrophy and increase may reflect a need for more

energy derived from oxidative phosphorylation.

This metabolic route requires oxygen, and consequently,

many more mitochondria are needed. However, mitochon-

drial increase could also underlie muscle degeneration as

mitochondrial proliferation has been linked to apoptosis in

some myopathies [38]. Mitochondria experience frequent

fission and fusion to regulate their number, morphology,

and function. Bossy-Wetzel et al. [39] have proposed that a

balance between mitochondrial fission and fusion events is

required for normal mitochondrial function; excessive

mitochondrial fission results in a mitochondrial malfunc-

tioning, which increases reactive oxygen species [40].

Muscle disorganization

In our experiments, we observed changes in the organiza-

tion of the sarcomere. In particular, Z lines appeared

disorganized, and M bands were sometimes absent. Z lines

are important structures in striated muscle fibers, which

provide stability and functional integration of the sarco-

mere. Additionally, Z lines play an important role in

myofibril assembly and in anchoring muscle filaments [41,

42]. M bands are composed of different proteins [43] and

glycolytic enzymes [42], which help to stabilize the sar-

comere. In muscle pathologies, sarcomeric proteins can

also be disorganized [44]; therefore, it is possible that the

modifications we observed in the sarcomere of 3-NP-trea-

ted muscles were due to the damage of structural proteins

in Z lines and M bands. Further experiments are necessary

to determine changes in the expression and assembly of

different sarcomeric proteins when oxidative metabolism is

uncoupled.

Underlying mechanism to muscle changes

Mitochondrial failure and oxidative stress activation seem

to underlie the enzymatic changes, muscle disorganization,

and mitochondrial increase observed in our study. To

corroborate the presence of oxidative stress, NO and LPO

levels were evaluated, confirming that mitochondrial mal-

functioning results in oxidative stress and ROS production

as previously suggested [40].

Behavioral changes and striatal damage

In previous reports, it has been described that 3-NP-treated

mice exhibit a progressive development of several behav-

ioral changes and alterations in motor activities, including

orofacial dyskinesias, increase in grooming, and cognitive

impairments [45, 46]. Using chronic administration of 3-NP

in low doses, we found a reduction in the latency to clasp

and abnormal clasping of the hind limbs and forelimbs after

being suspended by the tail. These results are in agreement

with observations made in transgenic HD mice [47].

Besides muscle alteration, striatal tissue from 3-NP-

treated mice exhibited an increase in damaged cells

(Fig. 7) and a reduction in calbindin positive neurons (data

not yet published). In transgenic HD mice, the develop-

ment of behavioral symptoms correlates with a medium

spiny neuron reduction in the striatum, identified by cal-

bindin [47]. We conclude that low doses of 3-NP also

affects striatal cells but to a lesser degree.

Functional implications of 3-NP effects at the periphery

Using systemic (i.p.), low doses of 3-NP, the present study

provided an opportunity to test the functional significance

of a reduced mitochondrial complex not just at the fore-

brain but also at the peripheral level. This approach showed

that inhibition of oxidative phosphorylation induced

252 Neurol Sci (2011) 32:241–254

123

changes in muscle enzymatic activity and ultrastructure. 3-

NP has been used as a model to study HD. If this mito-

chondrial toxin really imitates the neuropathological

changes observed in patients afflicted with HD, it is plau-

sible that abnormal mitochondrial function in non-neural

tissue during first stages of the disease may be responsible

for the body weight loss observed in patients afflicted with

the illness as a result of muscle damage [12, 15]. It is

interesting that experiments using uncoupling proteins of

oxidative phosphorylation, such as UCP proteins and

dinitrophenol, also produce a loss of weight in experi-

mental animals [48].

Although the cause of weight loss remains unclear in

several pathologies, it is possible that changes such as the

ones described in the present study could exacerbate

metabolic activity during the early stages of neurological

illnesses, producing high metabolic demands to counteract the

bioenergetics failure [49]. All these changes may generate

an increase in the energy consumed by the muscles; this

hypothesis has been suggested for many of the diabetogenic

neurodegenerative disorders in which alterations in oxida-

tive phosphorylation and mitochondrial metabolism are

compromised [50]. Also, in models of food restriction or

malnutrition, an animal’s size and body weight are most

affected to preserve brain functions [51, 52].

Our experimental findings could have implications for

the early clinical management of diseases in which mito-

chondrial failure is presumed because muscle atrophy

could conceivably exacerbate the pathological process.

Ongoing work in our laboratory is designed to further

characterize why relatively low insults in oxidative phos-

phorylation lead to brain and muscle atrophy in an animal

model of HD.

Acknowledgments We thank Dr. P. Maldonado for processing

muscle samples for LPO, Dr. I. Terrazas for his lab support in NO

evaluation, H. Barrera M Sc for his help in image acquisition and the

reviewers whose comments improved greatly the paper. This work

was supported by Consejo Nacional de Ciencia y Tecnologıa 42598

Grant, Programa de Apoyo a Proyectos de Investigacion e Innovacion

Tecnologica; IN209103 and IN201307 Grants.

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