https://doi.org/10.33549/physiolres.932348

Physiol. Res. 62: 119-123, 2013

SHORT COMMUNICATION

Hindlimb Suspension Does Not Influence Mechanical Sensitivity,Epidermal Thickness, and Peripheral Nerve Density in the Glabrous
Skin of the Rat Hind Paw
Y. TANAKA1, J. NAKANO2, Y. HAMAUE3, Y. SEKINO3, J. SAKAMOTO4, H. KATAOKA3,M. OKITA3
1

Department of Rehabilitation, Nagasaki Memorial Hospital, Nagasaki-shi, Japan, 2Unit of Physical
Therapy and Occupational Therapy Sciences, Nagasaki University Graduate School of Biochemical
Sciences, Nagasaki-shi, Japan, 3Department of Locomotive Rehabilitation Science, Unit ofRehabilitation Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasakishi, Japan, 4Department of Rehabilitation, Nagasaki University Hospital, Nagasaki-shi, Japan
Received March 6, 2012
Accepted September 24, 2012
On-line November 22, 2012

Summary

Corresponding author

Our aim was to investigate the influence of microgravity on the

Jiro Nakano, Unit of Physical Therapy and Occupational Therapy

sensitivity of the skin to mechanical stimulation, epidermalSciences, Nagasaki University Graduate School of Biochemicalthickness, peripheral nerve density in the upper dermis, andSciences,serum levels of a stress marker in a rat hindlimb suspension (HS)Fax: +81-95-819-7919. E-mail: nakano-j@nagasaki-u.ac.jp

1-7-1

Sakamoto,

Nagasaki-shi

852-8520,

Japan.

model. Thirty 8-week-old male Wistar rats were randomly divided
into 3 groups: HS, n=10; sham HS, n=10; control, n=10. The
suspension system was attached to rat tails in both the HS andsham-HS groups, but the hindlimbs were suspended only in theHS group. The HS and sham-HS groups were treated for
4 weeks. In behavioral tests using von-Frey filaments (n=5 ineach group), mechanical hypersensitivity developed in the HSand sham HS groups. Serum corticosterone levels increased
significantly in the HS and sham HS groups compared to thecontrol group, and no changes in epidermal thickness or
peripheral nerve density were observed immediately after theremoval of HS (n=5 in each group). These data indicated thatthe mechanical hypersensitivity observed in the HS group was
not caused by microgravity or inactivity, but rather by restraintstress. We suggest that microgravity does not affect skinsensitivity and histology in these animals.
Key words
Hindlimb suspension • Mechanical hypersensitivity • Epidermal
thickness • Peripheral nerve • Stress

Hindlimb suspension (HS) animal models arewidely used in studies investigating the effect of
microgravity and spaceflight on the locomotorium suchas bone and muscle (Templeton et al. 1984). Changes inthe viscera (Stein et al. 2005), circulation (De Salvatore
et al. 2004), and blood vessels (Fujino et al. 2005) havealso been studied using the HS model. A few
histochemical studies that investigated muscularsensation in response to HS showed no effect on muscle
spindles or the size and shape of Golgi tendon organs(Asmussen and Soukup 1991). However, extremely few
studies have addressed the changes in skin tissues andperipheral nerves or the sensory aspects of suspension
hypokinesia. Changes in skin tissue and peripheral nerves
may affect the skin sensation (Li et al. 2009, Nakano etal. 2012). Although a few reports showed thatmicrogravity and non-weight bearing are associated withpressure hypersensitivity in rats, the effect of stress was
not examined using a sham group in these studies

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120 Tanaka et al.
(Chowdhury et al. 2011, Kauppila et al. 1998). It is wellknown that various stresses can induce hypersensitivity(Bardin et al. 2009, Imbe et al. 2004, Nasu et al. 2009).
In studies using HS model rats, the influence of restraint
stress should not be ignored because HS affects thewellness of the animal. A previous study showed
increased serum levels of stress markers such as
epinephrine in HS model rats (Kawano et al. 1995). Thus,
whether hypersensitivity is a specific consequence ofmicrogravity, or if other stimuli can also cause it, remains
unclear. To address these issues, we examined thechanges in the sensitivity of the skin in response tomechanical stimuli, epidermal thickness, peripheral nervedensity in the upper dermis, and serum levels of a stressmarker by using HS model rats.
This study was approved by the Ethics Review
Committee for Animal Experimentation of Nagasaki
University. Thirty 8-week-old male Wistar rats weighing
220±18 g from Kudo Laboratories (Tokyo, Japan) wererandomly divided into control, HS, and sham-HS groups(n=10 each). Rats were housed individually in 30 × 40 ×20 cm cages at 24 °C, with water and food available adlibitum. The suspension system was attached to the tail ofrats in the HS group, and the height of the system wasadjusted so that the suspended hindlimbs did not touch
the floor (Templeton et al. 1984). The same suspension
system was attached to the tails of rats in the sham-HSgroup but the hindlimbs were not suspended, so the rats
could move normally in their cages. All other aspects ofthe set-up in the three groups were identical. The periodof HS was 4 weeks.
Behavioral tests to assess bilateral hindlimb
mechanical sensitivity were performed weekly in 5 rats
from each group. Rats were placed in a small animal
fixator in a quiet room during the behavioral test. Theglabrous skin of the hind paw was probed 10 times with avon Frey filament (15 g; North Coast Medical, USA)
repeatedly, and the paw-withdrawal response (PWR) wasdetermined. The body weights of rats in the 3 groups
were also measured weekly. At the end of the HS period,
the suspension system was removed in the HS and shamHS groups. The rats that underwent behavioral testing
were maintained in a normal breeding state, and thebehavioral tests and weight measurements were extended
for 8 weeks after the removal of HS. The other 5 rats ineach group that were not subjected to behavioral tests
were intraperitoneally anesthetized with sodium
pentobarbital (40 µg/g) immediately after the removal ofHS, and blood was collected by venipuncture of the tail.

Vol. 62
The serum was extracted after clotting and centrifugationof blood, and it was stored in the freezer. Subsequently,transcardial perfusion was performed with 4 %paraformaldehyde in 0.1 M phosphate buffer, and theglabrous skin of the hind paw was removed bilaterally.
The tissues were embedded in tragacanth gum aftersoaking in 10 % saccharose and stored at −80 °C. Serum
corticosterone levels were estimated as a stress marker
using the Corticosterone EIA kit (YK240; YanaiharaInstitute Inc., Japan) (Hong et al. 2011). Although theinfluence of pentobarbital anesthesia cannot not beignored in the determination of serum corticosterone
levels (Green and Moor 1977), its effect on the results
was minimized by employing uniform anesthesia
conditions for all rats during blood sampling. Frozen skinsections (10 μm) were cut using a cryostat (CM1950;Leica, Germany) and stained with hematoxylin-eosin.
Epidermal thickness was measured using the Scion Image
software (Scion Corp., USA) at 7 random points perimage (×200). Five sections per skin sample were usedfor measurements. Immunohistochemical staining for
peripheral nerves was performed on frozen sections(30 μm) with rabbit anti-PGP9.5 antibody (1:2000;
Calbiochem, Germany), as previously described (Lauriaet al. 2005). For semiquantitative analysis, the number of
PGP9.5-positive nerve fiber fragments in the upperdermis (0-50 μm in depth under the epidermis) wascounted on the images (×200) obtained from 5 sections
per skin sample and classified according to the skin tissue
width. The number of PGP9.5 nerve fibers per 1 mm-unit
was calculated as “peripheral nerve profiles.” Data arepresented as mean ± SEM. Statistical analysis wasperformed using ANOVA with the Scheffe post-hoc test,and P<0.05 was considered significant.
Although body weight increased at all time
points in the HS, sham-HS, and control groups, weightgain in the HS and sham-HS groups was lower than thatin the control group (Fig. 1A). The PWR increased
significantly during HS in the HS and sham-HS groups
compared to the control group (P<0.01), and this increasein the PWR persisted for 5 and 6 weeks after removal of
HS (Fig. 1B). However, no significant differences inbody weight and PWR were observed between the HS
and sham-HS groups. Serum corticosterone levels
increased significantly in the HS and sham-HS groups
compared to the control group (P<0.01) (Fig. 2A), withno significant differences in corticosterone levels
between these 2 groups. In the histological analysis, nopathological changes such as inflammation were2013

Mechanical Sensitivity in Hindlimb-Suspended Rats

121

observed in skin tissue. No significant changes inepidermal thickness or peripheral nerve profiles in theglabrous skin of the hind paw were observed in any of thegroups (Fig. 2B,C).

A

B

Body weight (g)ControlHSsham-HS

550
500
450
400350*300*2500*
*

**
*
*

*

12**
*

*
After removal of HS

HS0*3456 7weeks

8Paw-withdrawal Response (%)

100600ControlHSsham-HS

*60*
*

*20**

*40*

*01234*
*

*

*

*0

9 10 11 12After removal of HSHS805 6 7weeks**89 10 11 12Fig. 1. Changes in body weight (A) and mechanical sensitivity (B) during and after HS. The HS and sham-HS procedures resulted in asignificant decrease in weight gain; no significant difference was observed between the HS and sham-HS groups (A). The PWR inducedby 15-g von Frey filaments significantly increased in the HS and sham-HS groups during HS compared to the control group; nosignificant difference was observed between the HS and sham-HS groups (B). HS: hindlimb suspension. * vs. control group, P<0.05.

400

*300

*200
1000Control

HSsham-HS

C7060
50403020100Control

HSsham-HS

Number of PGP9.5-positive fibers
in the upper dermis ( / unit width)

B
Epidermal Thickness (mm)Serum corticosterone levels (ng/ml)

A

50403020
100Control

HSsham-HSFig. 2. Changes in serum corticosterone levels (A), epidermal thickness (B), and peripheral nerve density in the upper dermis (C)immediately after the removal of HS. Serum corticosterone levels significantly increased in the HS and sham-HS groups compared tothe control group; no significant difference was observed between the HS and sham-HS groups. The procedures performed in the HS
and sham-HS groups did not cause changes in epidermal thickness and density of PGP9.5-positive nerve fibers in the upper dermis.
* vs. control group, P<0.05.

No differences were observed between the HS
and sham-HS groups in any of the parameters tested.
Chowdhury et al. (2011) suggested that non-weightbearing conditions in HS model rats may lead to pressure
hypersensitivity. In the present study, although the same
HS model was used, mechanical hypersensitivity wasobserved in the sham-HS group as well. Rats in the shamHS group were attached to the trolley suspension systemin the same manner as those in the HS group during HS,
and they were allowed to bear weight on their hindlimbs

while walking. Our results indicate that the mechanicalhypersensitivity observed during HS cannot be attributed
to the effects of microgravity. In addition, changes in
epidermal thickness and peripheral nerve density, which
are often observed in diabetes and neuropathy (Chao etal. 2011, Kojundzic et al. 2008, Lauria et al. 2005,
Romanovsky et al. 2010), were not detected in the HSgroup. These results indicate that the hypersensitivity in
the HS group was not due to histological changes of
peripheral tissues such as skin and nerve. Because a122 Tanaka et al.
similar increase in the serum levels of corticosterone,
which was used as a stress marker, was observed in theHS and sham groups, it is possible that mechanicalhypersensitivity in these groups was induced by stress.
Body weight analysis confirmed the involvement ofstress. Attachment of the suspension system may haveresulted in the development of restraint stress andpossibly mechanical hypersensitivity (Bardin et al. 2009,Imbe et al. 2004, Nasu et al. 2009).
Inactivity and immobilization of extremities,
such as those associated with casting after fracture, havebeen reported to induce hypersensitivity (Allen et al.
1999, Guo et al. 2004). Terkelsen et al. (2008) conducted
an experiment involving healthy human subjects in which
the forearm was immobilized by casting for 4 weeks;subsequently, transient movement-provoked pain andincreased skin temperature in concert with mechanicaland cold hyperalgesia were induced in distal parts of theimmobilized extremity. Although the detailed
mechanisms are unclear, several factors have been
associated with the induction of hypersensitivity,
including hypokinesia; reduction of the sensory input;changes in peripheral tissue, including the skin; blood

Vol. 62
flow deficiencies; central neuron system changes; andstress. In our HS model, and considering the results of thestudy, hypokinesia, reduction of sensory input, andchanges in the skin tissue can be excluded. However,stress may have affected the results of various
parameters, and further investigation will be necessary in
the future.
In conclusion, mechanical hypersensitivity
observed in a HS model rat was related to stress but notto microgravity. In addition, our results indicated thatmicrogravity did not affect the sensitivity or histology ofthe glabrous skin.

Conflict of Interest
There is no conflict of interest.

Acknowledgements
This study was supported by a grant-in-aid for scientificresearch (No. 23500587) from the Japan Society for thePromotion of Science. The authors thank Takuya
Fukushima (Department of Rehabilitation, Shimonoseki
City Central Hospital, Shimonoseki-shi, Japan) for his
assistance in the experimental work.

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