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3. Results
3.1. Assessment of general toxicity
In the five Taxol -injection group, two rats died, one
after the first and one after the fourth Taxol injection,
after a substantial decrease in body weight and piloerection were observed. All other animals survived until the end of
the study and received the five injections as scheduled in the protocol.
A significantly lower body weight gain was observed from the fourth injection to the end of the study 215
compared to both control groups; P,0.01 in the multiple
Taxol -treated group. Therefore, these rats showed a lower decrease in motor activity, however this was not significant
compared to the control groups. No difference in cutaneous temperature was noted in the treated rats and both control
groups.
There was no mortality in the single Taxol injection
groups. A significant decrease in body weight maximal decrease: 217, P,0.001 and 210, P,0.01 after 3
days, for 32 and 16 mg kg, respectively was observed,
Fig. 1. Mechanical hyperalgesia five injections — time-course of pain
with a total recovery within 2 weeks after the injection.
withdrawal thresholds in treated y, Taxol 16 mg kg, n59 and control
d, saline, n59 m, vehicle, n59 rats submitted to an increasing
No rat in the control groups died throughout the course
pressure on the hind paw. Each rat received one i.p. injection arrow per
of the experiment and no alterations in motor activity,
week for 5 weeks. Scores were determined before each injection. The
cutaneous temperature or clinical signs were observed.
bars represent S.E.M. A significant difference P,0.05, P,0.01;
Body weight gain was similar for both saline and vehicle
analysis of variance followed by Student’s t-test in unpaired series was
groups.
observed versus the saline group from the first injection to 7 days after
the last injection for the Taxol 16 mg kg group, and from the 7th to the 21st days after the last injection for the vehicle group.
3.2. Behavioural examinations 3.2.1. Grip strength test
Whatever the treatment, the dose and the schedule of were lower than in the Taxol
group for the last injection
administration, we observed a normal increase in the motor 214.0610.4 g and 230.6620.5 g for vehicle and Taxol ,
force without any significative difference compared to respectively. No modification of the vocalisation thres-
saline control group. holds was observed at any time in saline-treated group.
In the single Taxol -injection group, vocalisation thres- 3.2.2. Randall and Selitto test
holds decreased both in the 16 and 32 mg kg groups, from In all experiments, mean vocalisation thresholds were
the 4th to the 9th day 16 mg kg and to the 14th day 32 not significantly different between control and drug-treated
mg kg after the injection, with a maximal decrease on the groups before the first injection.
7th day after injection, i.e., 225 P,0.01; in 80 of
In the multiple Taxol -injection group, a significant rats and 230 P,0.01; in 90 of rats, respectively. No
decrease in vocalisation threshold 80 of rats compared decrease in vocalization threshold was observed in the
to the baseline and control groups, was observed from the control groups during the course of the experiment Fig.
first to the fifth injection P,0.01 Fig. 1. The maximum 2.
decrease in vocalisation threshold maximal decrease: 233, P,0.01 was observed after the fifth injection.
3.2.3. Von Frey hairs test From 2 weeks after the fifth injection, vocalisation thres-
A significant reduction P,0.01 in the withdrawal holds increased progressively but up to 21 days after the
threshold to von Frey filaments 287 and 281 com- last injection remained significantly lower to baseline
pared to baseline values was observed in two rats 14
values P,0.05. repeatedly treated with Taxol
from the fourth injection to In the multiple vehicle-injection group, a decrease in
the end of the experiment. No significant difference in vocalisation threshold compared to baseline values 57
mean withdrawal threshold was observed in treated rats of rats was noted from the fifth injection to the end of the
compared to control groups.
study maximal decrease: 223, P,0.05, 14 days after In the Taxol
single injection group, at either 16 or 32 the last injection. Moreover, the vocalisation thresholds
mg kg, thresholds were not significantly modified. In both
N . Authier et al. Brain Research 887 2000 239 –249
243
Fig. 3. Thermal hypoalgesia five injections — paw withdrawal latencies to a radiant heat stimulus applied to the plantar surface of the hindpaw of
Fig. 2. Mechanical hyperalgesia one injection — time-course of pain
treated y, Taxol
16 mg kg, n59 and control d, saline, n59 m,
withdrawal thresholds in treated y, Taxol 16 mg kg, n59 h, Taxol
vehicle, n59 rats. Each rat received one i.p. injection arrow per week 32 mg kg, n59 and control d, saline, n59 m, vehicle, n59 rats
for 5 weeks. Scores were determined before each injection. The bars submitted to increasing pressure on a hind paw. Each rat received one i.p.
represent S.E.M. A significant difference P,0.05, P,0.01, analysis injection arrow. Scores were determined on days 2, 4, 7, 9 and 14 after
of variance followed by Student’s t-test in unpaired series was observed injection. The bars represent S.E.M. A significant difference P,0.05,
versus control groups from the 2nd to the 5th injection for the Taxol 16
P,0.01; analysis of variance followed by Student’s t-test in unpaired mg kg group.
series was observed versus control groups from days 4 to 9 for the
Taxol 16 mg kg group, and from days 4 to 14 for the Taxol 32 mg kg.
control groups, withdrawal thresholds were not decreased at any time.
3.2.4. Plantar test
In repeated Taxol -injected rats, compared to control groups, significant increases in paw withdrawal times from
the second injection P,0.01 to 7 days after the last injection P,0.05, were observed. The maximal increase
was 131 compared to baseline P,0.01. This thermal hypoalgesia affected 57 of treated rats Fig. 3.
After one single Taxol injection at 32 mg kg, com-
pared to control groups, a significant increase in withdraw- al latency threshold was noted 4 and 7 days after the
injection, with a maximum of 117 on day 7 P,0.05. No modification was noted after administration of 16
mg kg Fig. 4.
Whatever the administration schedule, there was no alteration in the withdrawal latencies of the control groups.
Fig. 4. Thermal hypoalgesia one injection — paw withdrawal latencies to a radiant heat stimulus applied to the plantar surface of the hindpaw of
treated y, Taxol
16 mg kg, n59 h, Taxol 32 mg kg, n59 and
3.2.5. Tail immersion test
control d, saline, n59 m, vehicle, n59 rats. Each rat has received
When thermal allodynia was assessed, tail withdrawal
one i.p. injection arrow. Scores were determined on days 2, 4, 7, 9 and
times did not increase at any time in either the control or
14. The bars represent S.E.M. A significant difference analysis of
the Taxol -treated rats, whatever the schedule of adminis-
variance followed by Student’s t-test in unpaired series was observed
tration.
versus control groups on days 4 and 7 for the Taxol 32 mg kg group.
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difference was observed between the Taxol and the
control groups. 3.4. Morphological examination
Results of the morphological examination light and electron microscopy are summarised in Table 1.
3.4.1. Light microscopy Degenerative changes were observed on both paraffin
and semithin sections, in the sciatic nerve, the nerve fibres in the paw subcutaneous tissue, and in the lumbar spinal
cord. In the five injection Taxol or vehicle groups, at the
7 or 21 day necropsies after the last intraperitoneal injection, lesions were found in all organs examined. In the
single Taxol injection group, a few changes were found
in the subcutaneous nerves 16 and 32 mg kg and in the sciatic nerve 32 mg kg. In the single vehicle injection
group, and in all saline control groups, no lesion could be
detected. In the five injection Taxol or vehicle groups no
Fig. 5. Peripheral nerve conduction velocity decrease — sciatic nerve
major differences could be found between the Taxol or
conduction velocity in paclitaxel-treated h, Taxol 16 mg kg, n56 and
the vehicle-treated groups, and between the 7 versus 21
control j, saline, n56 9, vehicle, n56 rats measured after five
days post-injection groups.
injections and 21 days after the last injection. Each rat received one i.p. injection arrow per week for 5 weeks. The bars represent S.E.M. For the
Taxol 16 mg kg group, a significant difference P,0.05; analysis of
3.4.1.1. Sciatic nerve Figs. 6 and 7. In the longitudinal
variance followed by Student’s t-test in unpaired series was observed
paraffin sections rare Wallerian degeneration and axonal
versus the control groups 7 days after the 5th injection.
swellings giant axons were observed. In Epon semithin sections, degenerative changes were obvious, with a zonal
3.3. Electrophysiological study pattern in the most severe cases. In the affected area, in
myelinated fibres identified as large diameter fibres, the
Taxol induced a significant decrease P,0.05 in
axonal clear centre of the fibre was replaced by dark peripheral nerve conduction velocity compared to the
myelinic debris, as confirmed by electron microscopy. control groups, 7 days after the last injection. Three weeks
after the end of the last injection, differences between 3.4.1.2. Spinal cord. Degenerative changes, similar to the
treated and control groups were no longer apparent Fig. lesions described above in the sciatic nerve, were ob-
5. Seven days after the single injection, no significant served, scattered in the white matter of the lumbar spinal
Table 1
a
Summary of histological examinations after one or five Taxol injections in rats
Nerve of paw subcutaneous tissue Sciatic nerve
Lumbar spinal cord white matter
Taxol 5316 mg kg 111
11 1
Sac 7 days PI Ax; Myel; Schwann
Ax; Myel Ax; Myel
Vehicle 5 injections 111
11 1
Sac 7 days PI Ax; Myel; Schwann
Ax; Myel Ax; Myel
Taxol 5316 mg kg 111
11 1
Sac 21 days PI Ax; Myel; Schwann
Ax; Myel Ax; Myel
Vehicle 5 injections 111
11 1
Sac 21 days PI Ax; Myel; Schwann
Ax; Myel Ax; Myel
Taxol 1332 mg kg 1
1 1
Sac 7 days PI Ax
Ax Ax
Taxol 1316 mg kg 1
Sac 7 days PI Ax
Vehicle 1 injection Sac 7 days PI
a
0, no detectable change; 1 to 111, degenerative changes as described in text, graded from the less to the most severe. Sac, rats sacrificed 7 or 21 days after the last intraperitoneal injection. Ax; Myel; Schwann, axons, myelin sheaths and or non-myelinated Schwann cells were affected in the group.
N . Authier et al. Brain Research 887 2000 239 –249
245
Fig. 6. Swollen degenerated axon — longitudinal paraffin section of the Fig. 8. Degenerated subcutaneous fibres — semithin transversal section
sciatic nerve from a Taxol -treated rat five injections at 16 mg kg of fine subcutaneous nerve fibres in the paw of a Taxol -treated rat five
showing a swollen degenerated axon arrow. Haematoxylin and Eosin injections at 16 mg kg showing numerous degenerative lesions arrows.
3300. Toluidine Blue 31200.
cord. Neurons in the gray matter remained unaffected in all the light microscope, there were no major ultrastructural
groups.
differences between the Taxol and the vehicle-treated
groups. The most conspicuous changes were found in the 3.4.1.3. Subcutaneous tissue Fig. 8. In the fine subcuta-
paw subcutaneous nerve fibres. neous nerves from the paws, degenerative changes affected
almost all nerve fibres, including those of both large and 3.5.1. Subcutaneous nerve fibres Figs. 9 and 10
small diameter. Lesions were similar to those noted for the Axons were degenerated. They were darkened, with
sciatic nerve, and phagocytosis of degenerative myelinic organelle accumulation, mostly neurotubules and or vesi-
debris was clearly detected at high magnification. cles, and often seemed compressed by Schwann cells
changes as described below. Vesicles were empty or filled 3.5. Ultrastructure
with lamellar debris. Most myelinated and a few non- myelinated axons were affected.
At the ultrastructural level, axonal and or Schwann cells
Schwann cells and myelin sheaths: vacuolation of changes were found in all Taxol or vehicle groups, except
enlarged abaxonal and adaxonal parts of the Schwann cells in the single injection vehicle group. No changes were
was frequent, with accumulation of various debris, giving noted in saline control groups.
the axon an angular shape. In the most severe cases, In the Taxol and vehicle five injection groups, as under
myelin sheaths were also affected by partial delamination of collapsed myelin sheaths ovoids or complete destruc-
Fig. 7. Degenerated large fibres — semithin transversal section of the Fig. 9. Normal subcutaneous fibres — ultrathin section of fine subcuta-
sciatic nerve from a Taxol -treated rat five injections at 16 mg kg neous nerve fibres in the paw from a control saline rat showing several
showing numerous degenerated large nerve fibres arrows. Toluidine normal myelinated arrows and non-myelinated arrowheads axons.
Blue 31200. Uranyl acetate and lead citrate 35000.
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Fig. 10. Degenerative subcutaneous nervous lesions — ultrathin section Fig. 12. Axonal degenerative changes — ultrathin section of fine
of fine subcutaneous nerve fibres in the paw of a Taxol -treated rat five subcutaneous nerve fibres in the paw of a Taxol -treated rat a single
injections at 16 mg kg showing several degenerated myelinated axons injection at 32 mg kg showing axonal degenerative changes stars while
arrows, beside few normal nerve fibres star. Degenerative changes are myelin sheaths remain within normal limits arrows. Uranyl acetate and
also found in non-myelinated schwann cells arrowheads. Uranyl acetate lead citrate 38700.
and lead citrate 35000.
organelles and vacuoles in the sciatic nerve 32 mg kg and subcutaneous tissue 16 and 32 mg kg. Schwann
tion and
subsequent phagocytosis.
Non-myelinated cells and myelin sheaths remained normal and no change
Schwann cells were also affected and showed various was detected in the spinal white matter. No ultrastructural
degrees of degeneration, mostly vacuolisation. change was found in the corresponding vehicle group Fig.
13. 3.5.2. Sciatic nerve Fig. 11 and spinal cord
In the affected areas of transverse sections of the sciatic nerve, the nervous fibres showed the various degrees of
alteration described above for subcutaneous fibres, with the
4. Discussion
Kategori