Mechanical Withdrawal Thresholds in Normal and Neuropathic Rats are Affected by Probe Material

Abstract

Aim of investigation: Mechanical sensitivity in rats is usually tested using nylon filaments of different diameters. With an electronic pressure algometer (Ängeby Möller et al, J. Neurosci. Methods 84 (1998) 41-47) we have shown that withdrawal thresholds increase with increasing nylon probe diameter (Ängeby Möller and Berge, Abstracts 9th World Congress on Pain, 1999:14). The aim of the present study was to investigate the effect of probe material and size on mechanically induced withdrawal responses. Material and methods: Two mm of the L5 spinal nerve distal to the dorsal root ganglion was removed on the left side to prepare male Sprague-Dawley rats, weighing 150-200 g at the start of the experiments, with a surgical neuropathy (Kinnman and Levine, Neuroscience 64 (1995) 751-67). Control groups were sham operated. In the first experiment, withdrawal thresholds were assessed in a group of untreated test-naive rats, using probes made from nylon or stainless steel ( = 1 mm) in a crossover design. In a second experiment, mechanical withdrawal thresholds were measured in a separate group of rats using steel probes of four different diameters (0.2, 0.3, 0.5 and 1.0 mm). In addition, the withdrawal response types were scored. Thresholds were measured and responses were scored before and up to two weeks after surgery. Results and conclusion: Untreated rats showed withdrawal thresholds from nylon and stainless steel probes with 1 mm diameter at 42.9 ± 1.3 and 34.8 ± 1.2 g respectively. As previously shown for nylon probes (Ängeby Möller and Berge, Abstracts 9th World Congress on Pain, 1999:14), reducing the diameter of the steel probe resulted in lower withdrawal thresholds. The withdrawal response type changed from mainly brisk withdrawal with the 0.2 mm steel probe to a higher frequency of passive lifting before withdrawal by the 1.0 mm probe. After operation, the lesioned side was withdrawn at lower thresholds, tested with all sizes of steel probes, compared to the non-operated paw and both sides of the control group. Compared to testing with nylon probes, the same diameters of steel probes resulted in slightly lower withdrawal thresholds and a tendency to higher response score (more vigorous withdrawal) at all times. In conclusion, stainless steel probes, which are mechanically more stable than nylon probes, can be used for assessing mechanical withdrawal thresholds in rats. The results confirm previous data showing that probe size influences the withdrawal response to mechanical stimulation. In addition, the present results confirm that the threshold difference between probe sizes is more correlated to the tension induced at the edge of the probe than the pressure by the probe area.

Full text

Stainless steel
Probe diameter (mm)
0.2 0.3 0.5 1
Threshold (N)
0.1
0.2
0.4
L5 lesion/ipsilateral
L5 lesion/contralateral
Sham/left
Sham/right
Stainless steel
0.2 0.3 0.5 1
Threshold (N)
0.1
0.2
0.4
Nylon
Probe diameter (mm)
0.25 0.5 1
Threshold (N)
0.1
0.2
0.4
1 2
Threshold (N)
0.1
0.2
0.4
Metal Nylon
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MECHANICAL WITHDRAWAL THRESHOLDS IN NORMAL AND
NEUROPATHIC RATS ARE AFFECTED BY PROBE MATERIAL
K. Ängeby Möller, O.-G. Berge, Bioscience, AstraZeneca R&D Södertälje, S-141 57 Huddinge, Sweden
Introduction
The aim of the present study was to investigate
the effect of probe material and size on
mechanically induced withdrawal responses in
rats. Von Frey filaments, made from nylon probes
with varying diameter and stiffness are commonly
used for assessing mechanical thresholds. They are
significantly affected by changes in temperature and
humidity, making it difficult to compare results from
different laboratories and investigators. Using an
electronic pressure algometer ¹ we have compared
nylon probes of different diameter (from a previous
study ³) to mechanically more stable stainless steel
probes. Measurements were made in untreated rats
as well as animals prepared with a surgical
neuropathy which reproducibly lowers withdrawal
thresholds.
Methods
Animals
Male Sprague-Dawley rats weighing 150-200 g at the time
of surgery were used.
Surgery
Under isoflurane anesthesia, 2 mm of the left L5 spinal
nerve distal to the dorsal root ganglion was removed
(method modified after Kim and Chung ²). A control group
was sham operated.
Apparatus
The pressure algometer consists of a force transducer with
an interchangeable nylon or stainless steel probe
connected to a computerized data collection system
allowing on-line display of the applied force. Data stored on
the computer can be retrieved for analysis.
Testing
In the first experiment untreated rats (n=12) were tested
with nylon and stainless steel probes ( = 1.0 mm) in a
crossover design. In the second experiment, lesioned
(n=16) and control (n=8) animals were tested using four
different metal probe diameters (0.2, 0.3, 0.5 and 1.0 mm)
in a crossover design. Thresholds were measured before
and up to two weeks after surgery. Data with nylon probes
(0.25, 0.5 and 1.0 mm) were taken from a previous study ³.
The experiments were approved by the appropriate
Medical Ethics Committee.
1. Probe material affects withdrawal threshold.
Untreated rats showed
withdrawal at significantly
(p<0.001) weaker stimuli when
tested with a stainless steel
compared to a nylon probe,
both with 1.0 mm diameter.
Mean ± SEM
2. Withdrawal thresholds increase with probe
size regardless of material.
Untreated rats measured
with metal probes of
different diameters
showed slightly lower
thresholds, compared to
testing with nylon probes
with the same diameters.
Mean ± SEM
3. Withdrawal response type changes with
probe size.
0.2 mm
1 2 3 4
0
20
40
60
0.3 mm
1 2 3 4
0
20
40
60
0.5 mm
1 2 3 4
Number of responses
0
20
40
60
1.0 mm
Withdrawal response type
1 2 3 4
0
20
40
60
0.25 mm
1 2 3 4
0
40
80
0.5 mm
1 2 3 4
Number of responses
0
40
80
1.0 mm
Withdrawal response type
1 2 3 4
0
40
80
Stainless steel
Nylon
Score Response type
1 Lifted by the probe
2 Lifted, then withdrawn
3 Rapidly withdrawn
4 Flinching / licking
The response in untreated rats
changed from mainly rapid
withdrawal with the
0.2 mm metal probe to more of
letting paw being lifted, then
withdrawn by the 1 mm probe.
Stainless steel probes resulted
in a tendency to more vigorous
withdrawal compared to testing
with nylon probes of the same
diameters.
4. Withdrawal thresholds in L5 lesioned rats differ with
probe size.
The paw of the lesioned side was
withdrawn at significantly lower pressure
compared to all other paws regardless of
probe size used.
5. Results are more consistent across metal probe size
when expressed as tension.
Pressure
Probe diameter (mm)
0.2 0.3 0.5 1.0
Threshold / area (N/mm2)
0.1
1
10 Tension
Probe diameter (mm)
0.2 0.3 0.5 1.0
Threshold / circumference (N/mm)
0.01
0.1
1
Thresholds, pooled from all values post surgery expressed in terms of pressure (force/area)
decreased more with increasing probe size, whereas thresholds expressed in terms of tension
(force/circumference) decreased less.
Lesioned rats: Circles
Sham-operated animals: Triangles
Non-operated side: Open symbols
Operated side: Filled symbols.
Mean ± SEM
Conclusions
Stainless steel probes can be used for assessing mechanical
withdrawal thresholds in rats.
Probe size, whether probes are made from stainless steel or nylon,
influences the withdrawal threshold and response type to mechanical
stimulation.
The present results confirm previous data that the threshold
difference between probe sizes depend more on the tension induced
by the probe edge than on the pressure exerted by the probe area.
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References
1. Ängeby Möller et al, J. Neurosci. Methods 84 (1998) 41-47
2. Kim, S. H. and Chung, J. M., Pain, 50 (1992) 355-363
3. Ängeby Möller and Berge, Abstracts 9th World Congress on Pain, 1999:14