Materials required:
- Thiopentone sodium (1 g reconstituted to 10 ml
or 100 mg/ml) and 1 ml tuberculin syringe.
- Dissection instruments: scissors, forceps (curved
artery forceps and toothed forceps), wooden
dissection board or rat dissection tray with four
side hooks for tying limb, glass dissecting probes
or glass seekers, dental scapula, and bone
rongeur.
- Unyielding-silk thread, warm paraffin wax or
mineral oil, 0.9% normal saline, droppers, cotton,
gauze pieces, cloth to pick up rat and gloves.
- Power Lab 26T with Lab Chart 8 software digital
data acquisition system, MLT0015 isotonic
transducer with a ML221 bridge amp, MLA270
stimulator cable (BNC to Micro-Hooks), MLA40
manipulator with stand (AD Instruments, New
South Wales, Australia), rod stand, pulley, pin
hook, T connector for pulley (if required),
connectors (for T connector and isotonic
transducer, if required), calibration weights (5 g, 10g and 15g etc.), and a ruler or measuring tape.
Animal:
A male or female Wistar rat of weight 200-250 g.
Objectives addressed:
- Define free loaded and after loaded conditions of
the muscle.
- Describe the effect of the above on the latent
period, contraction phase, relaxation phase and
height of contraction of simple muscle twitch.
- Explain the mechanism of difference in the work
done by the muscle under the two conditions.
Principle:
The effect of free loaded and after loaded condition
in isotonic mammalian skeletal muscle contraction
can be studied using in-situ nerve-muscle preparation
in rat which mimics physiological conditions
(intact blood supply with constant body temperature)
very closely unlike the in-vitro nerve-muscle
preparation regularly used for physiological practical
demonstration. Using isotonic transducer free
loading and after loading at different weights was
done in sciatic nerve-soleus muscle in-situ
preparation in an anaesthetized rat, and work done
is calculated in each condition from the data collected
and compared.
Experimental protocol:
Adult male Wistar rats (body weight 200-250 gm)
were obtained from institutional (All India Institute of
Medical Sciences, New Delhi, India) central animal
facility. The protocol was approved by Institute’s
Animal Ethics Committee (65/IAEC-1/2018). The
detailed procedure is described as follows:
Procedure:
- Calibration
The calibration of weight or force and displacement in the isotonic transducer as per the instruction of
the manufacturer (4).
- Animal preparation for the surgery
Acclimatize the rat to the departmental animal house
for one day with food (standard rat chow) and water
ad libitum. After one day, keep the rat on overnight
fast. Anaesthetize with thiopentone sodium
injected intraperitoneally with a dose of 50 mg/kg
body weight.
- Surgery and in-situ sciatic nerve-soleus muscle
preparation
Place the rat on a wooden dissection board and tie
all the limbs with thread to four corners. Skin one of
the legs from knee and down, dissect the soleus
muscle free from surrounding tissue. However, keep
the blood supply intake. Take utmost precaution not
to damage or put any drag on the blood supply of
the muscle.
Place a thread on the distal tendon of the soleus
muscle just proximal to its attachment to the
calcaneus. Release the distal attachment of the
muscle by cutting such that a small piece of
calcaneus is still attached to the Achilles tendon.
Tie the thread to isotonic transducer with the help of
a pulley system if need be so as to keep the
transducer in line with muscle-tendon unit, and the
pull of the muscle, with the soleus muscle parallel
to tibia bone. This ensures physiological movement.
Cut the gastrocnemius and plantaris tendons from
that of the soleus. Split the fascia between the soleus
and the gastrocnemius-plantaris about halfway up
the soleus. This should be, however, distal to the blood vessels which supply the belly of the soleus.
In this way, the movements of the gastrocnemius
and plantaris will not interfere with that of soleus
(5). Clamp the ankle and the middle part of tibia to
make the leg immobile and stable. The origin of the
muscle is immobilized (Fig. 1).
Identify the sciatic nerve and place stimulating
electrodes of the PowerLab 26T. Make sure the
soleus muscle temperature is around 37°C by pouring
pre-warmed 0.9% saline constantly over the epimysium of the muscle (6). Warmed Krebs-Ringer
solution may also be used (5). However, once the
setup is done, warmed paraffin or mineral oil may be
used instead by pouring it after making a container
with the loosened skin of the hind limb being pulled
around the sides of the exposed area of interest, so
as to minimize evaporative heat loss and distribute
heat evenly. A heat lamp may also be used. This is
done to keep the temperature as close to body
temperature as possible. However, make sure that
the core temperature of the animal does not exceed
38°C (7).
Instead of using the in-situ sciatic nerve-soleus
muscle preparation as above, one may use in-situ
preparation of gastrocnemius (8) or medial
gastrocnemius (6) or extensor digitorum longus (5)
or tibialis anterior (9) etc.
- Recording of muscle twitch under free loaded and
after loaded condition
Example of weights used may be 5 g, 10 g and 15 g.
The isotonic transducer is set in free loaded and
then in after loaded condition at different weights.
The sequence of free loaded and after loaded conditions may be randomized, or recordings are
taken in all possible sequences.
The stimulus given is a single square pulse of 1 ms
duration starting with 5 V, and gradually increased
(e.g., by 0.2V) to find maximal stimulus or maximal
voltage. A start delay of 10 ms, maximal repeat rate
of 1 Hz and amplifier range of 5 mV may be taken.
Different combinations, however, may be used as
per the requirement. A 15 ms pulse at 1 Hz, or 0.2 ms
pulse, or 50 µs pulse at 0.5V may also be used (5,
7). The sample frequency of 1 kHz or 2 kHz may be
used. The above stimulus parameters were chosen
after performing and recording muscle twitch using
the in-situ nerve-muscle preparation.
The recording of muscle twitch in free loaded and
after loaded conditions is done at supramaximal
stimulus. This is taken higher (eg: 0.5V higher) than
the maximal stimulus (5) or double the maximal
voltage etc (7). Rest duration of about 1-2 min may
be given between two successive stimuli (Fig. 2).
- Data analysis and work done calculation
Export the data to data pad of LabChart 8 and excel.
Record the maximum twitch amplitude or maximal
displacement. This is done for free loaded and
after loaded conditions at different weights. Calculate
the work done in both free loaded and after loaded
conditions by using this formula: work done in
ergs = weight (g) X maximum displacement (cm) X
981.
After the data collection, the rat is sacrificed with
the over dose of anesthetic or neck dislocation while
still under anesthesia.
