240 N
numbness and paraesthesia of the extremities [23,30,36], before administration to a final concentration of between
an increase in the vibratory threshold and the loss of deep 0.9 to 1.3 mg ml, depending on the animal weight and
tendon reflexes [30]. Nerve conduction velocities and ensuring that volumes of less than 5 ml would be injected
amplitudes of the sensory nerve action potentials are into the peritoneal cavity.
always reduced [11,23,30]. Moreover, there is no really effective therapy for the neuropathy-induced pain.
2.2.2. Control groups
Previous studies of Taxol -induced neuropathy in ani- The vehicle, made up of Cremophor EL Sigma, L’Isle
mals have described electrophysiological and histological d’Abeau, France and absolute ethanol Merck, Darmstadt,
changes [1,7–9,12] but none have fully described all types Germany in equal parts, was diluted at the time of
of nociception in animals after single dose or chronic injection with saline NaCl 0.9, in the same proportion
Taxol administration. The intraperitoneal route of ad-
as in the Taxol -treated groups. ministration, previously proposed by Apfel et al. [1],
Injected volumes of saline NaCl 0.9 were calculated Hamers et al. [18] and Cavaletti et al. [9] is particularly
according to the weight of the rat. suited to increasing the intratumoral disposition of the
active principle of Taxol , leading to high drug levels in 2.3. Experimental procedures
liver, colon, pancreas and ovaries. Previously we have
observed that five intraperitoneal injections, one per week, Taxol
was administered intraperitoneously according
do not modify the elimination kinetics of Taxol [13] and
to two different schedules:
are of too short a duration of administration to induce Taxol
multiple injection groups: one injection of 16
ascites [9]. Consequently, this schedule of administration mg kg Taxol per week for five consecutive weeks, giving
was used initially. a cumulative dose of 80 mg kg rat.
To fully describe the animal model of Taxol -induced Taxol
single injection groups: a single injection of 16
painful neuropathy in terms of pain, we have used the mg kg or 32 mg kg Taxol .
common battery of behavioural pain tests using noxious or non-noxious mechanical or thermal stimuli and the animal
2.4. Assessment of general toxicity strain commonly used for pain studies, i.e., the male
Sprague–Dawley rat. The schedule of administration was Body weights were measured before each injection,
adapted in order to preserve a satisfactory clinical status in every 3 days for 3 weeks after the fifth administration, and
the animals. In addition, electrophysiological and neuro- for 15 days following the single injection. Motor activity
pathological studies were performed using both light and was monitored every 4 days using an actimeter Actisystem
electron microscopy in order to describe possible changes Apelex, Passy, France. In addition, all rats were ex-
in nervous structures, to evaluate the functionality of the amined every day to detect clinical signs such as piloerec-
peripheral nervous fibres, and to determine the site of the tion or hindlimb weakness. Body temperature was assessed
neurotoxic action of Taxol . at regular intervals.
2.5. Behavioural assessment
2. Materials and methods
All behavioural tests were conducted before each in-
2.1. Animals jection of Taxol . The guidelines of the IASP Committee
for Research and Ethical Issues were followed for all Two hundred and fifty two male Sprague–Dawley rats
experiments [40]. `
Charles River, St-Aubin-Les-Elbeuf, France weighing 180 to 200 g at the beginning of the induction were used
2.5.1. Grip strength test [25] nine rats test dose and housed in plastic cages on a 12-h
After strain gauge was zeroed, the rat was placed with light 12-h dark cycle with access to water an food ad
both forepaws inside the front grip grid. When the rat libitum. Room temperature was maintained at 228C and
gripped the grid, it was steadily pulled backwards by the relative humidity was usually between 50 and 60.
tail until its grip was broken. Reading on strain gauge was Animals were allowed a 1-week acclimatisation period
recorded, the strain gauge was zeroed, and the rat retested before use in experiments.
until readings on three successive trials were obtained. 2.2. Drugs
2.5.2. Paw pressure test The paw pressure test has been previously described by
2.2.1. Taxol-treated groups Randall and Selitto [29]. Nociceptive thresholds, expressed
Paclitaxel Taxol , generously provided by Bristol– in grams, were measured by applying increasing pressure
Myers–Squibb, Paris, France, in vehicle see below, 5.9 to the right hind paw using an Ugo Basile analgesimeter
mg ml, was diluted in normal saline NaCl 0.9 just Apelex, Passy, France. The parameter used to quantify
N . Authier et al. Brain Research 887 2000 239 –249
241
the nociceptive threshold was the vocalization of the placed between two electrodes separated by 2.5 cm. After
animal. Rats were habituated to the testing procedures and estimating the depolarization threshold of the nerve, the
handling by the investigator during the week prior to the nerve was stimulated 50 times with a supramaximal
experiment. Experiments were performed until two similar stimulus. Nerve conduction velocity was directly calcu-
consecutive pressure values were obtained. The cut-off lated using a computer and a locally-made software. All
pressure was 450 g. measurements were performed under constant conditions
in a Faraday cage. 2.5.3. Von Frey hair test [10]
To assess changes in mechanical nociceptive thresholds, 2.7. Microscopic examination
rats were placed on a plastic mesh floor covered by transparent Plexiglas cages. On the week before the first
administration of Taxol or saline, rats were placed in the
2.7.1. Fixation of tissues test environment each day for 15 min. For testing, rats
At the time of sacrifice, based on satisfactory results of were allowed to acclimatise for 10 min. Successively
behavioural tests, selected rats were deeply anesthetized by greater diameter von Frey nylon monofilaments Stoelting,
an intraperitoneal injection of a 6 aqueous pentobarbital Wood Dale, IL, USA pressures 1.202, 1.479, 2.041,
solution 0.5 ml 100 g body weight. After opening the 3.630, 5.495, 8.511, 11.749, 15.136 and 28.840 g were
thoracic cavity, the vascular system was intracardiacally applied to the medial plantar surface of both hind paws.
perfused using a phosphate buffer solution pH57.2–7.4, For each filament, a series of five stimuli were applied
200 ml, followed by a 4 paraformaldehyde11 within a 2–3 s period per paw. Paw withdrawal threshold
glutaraldehyde 200 ml solution. was defined as the minimum pressure required to elicit a
Samples of the lumbar spinal cord, sciatic nerve and withdrawal reflex of the paw.
skin with the subcutaneous tissue of the paws were stored in the fixative solution as above, until trimmed for
2.5.4. Plantar test [19] histology and examination of the ultrastructure.
The rats were placed on a glass plate under a Plexiglas cage for 15 min before each experiment. On the week
before the first administration of Taxol or saline, rats
2.7.2. Histology were placed in the test environment each day for 15 min. A
Samples of lumbar spinal cord, sciatic nerve and skin radiant heat stimulus was applied to the plantar surface of
with subcutaneous tissue of the paws were paraffin embed- each hind paw by aiming a beam of light produced by a
ded, cut at 4 mm thick, and stained by the Haematoxylin 50-W projection lamp Apelex, Passy, France. When the
and Eosin method before examination using light micro- rat lifted the paw, the light beam was turned off auto-
scopy. matically allowing measurement of the time between the
start of the light stimulus and the foot lift. This time was defined as the withdrawal latency. Heat stimuli were
2.7.3. Electron microscopy stopped at 20.8 s to avoid skin injury. Three trials were
Blocks of lumbar spinal cord, sciatic nerve and skin with conducted on each hind paw, with 5 min between each
subcutaneous tissue of the paws of approximatively 2323 trial.
1 mm were postfixed in a 1 osmium tetroxide solution, and embedded in Epon after graded dehydration in alcohol.
2.5.5. Tail immersion test [27] Semithin and ultrathin sections were obtained using a
The tail of the rat was immersed in water maintained at Reichert Ultracut E ultramicrotome. Semithin sections
428C or 488C, until the tail was withdrawn. The duration of were stained with Toluidine Blue before light microscopy.
immersion was recorded and a cut-off time of 15 s was Ultrathin sections were contrasted by the uranyl acetate–
used. Rats were habituated to the testing procedures and to lead citrate method before examination with a Hitachi HU
handling by the investigator during the week prior to the 12A electron microscope at 75 KV.
experiment. 2.6. Electrophysiological study
2.8. Statistical analysis Five rats per batch at 7 and 21 days after the last
Treatments were randomised within each cage of six
injection of Taxol In the repeated Taxol -injection group
five injection study or four single injection study rats.
and one batch of five rats in the single Taxol -injection Behavioural data were evaluated using analysis of variance
group 7 days after injection were studied. The rats were Fisher test followed by a Student’s t-test to detect either
anaesthetised by intraperitoneal injection of ethyl carba- differences within treatment groups between baseline and
mate 2.5 mg kg. Samples of sciatic nerves were quickly post-injections values, or differences between each treat-
taken, preserved in a Ringer–Locke solution and then ment group on each day tested.
242 N
3. Results
