Physiological Characterization of Taxol- Induced Large-Fiber Sensory

Neuropathy in the Rat Kenneth D. Cliffer, PhD, Judith A. Siuciak, PhD, Susan R. Carson, BA, Heather E. Kadley, BS,

John S. Park, BS, Dacie R. Lewis, BS, Elizabeth Zlotchenko, MS, Trang Nguyen, BS, Karen Garcia, BS, James R. Tonra, PhD, Nancy Stambler, MS, Jesse M. Cedarbaum, MD, Sue C. Bodine, PhD,

Ronald M. Lindsay, PhD, and Peter S. DiStefano, PhD*

The cancer chemotherapeutic agent Taxol (paclitaxel) causes a dose-related peripheral neuropathy in humans. We pro- duced a dose-dependent large-fiber sensory neuropathy, without detrimental effects on general health, in mature rats by using two intravenous injections 3 days apart. Tests of other dosing schedules demonstrated the dependence of the severity of the neuropathy and of animal health on both the dose and the frequency of dosing. Pathologically, severe axonal degeneration and hypomyelination were observed in sections of dorsal roots, whereas ventral roots remained intact. Electrophysiologically, H-wave amplitudes in the hindlimb and amplitudes of predominantly sensory compound nerve action potentials in the tail were reduced. These effects persisted for at least 4 months after treatment. Motor amplitudes were not affected, but both motor and sensory conduction velocities decreased. The ability of rats to remain balanced on a narrow beam was impaired, indicating proprioceptive deficits. Muscle strength, measured by hindlimb and forelimb grip strength, and heat nociception, measured by tail-flick and hindlimb withdrawal tests, were not affected by Taxol. This model of Taxol-induced neuropathy in mature rats, with minimal effects on general health, parallels closely the clinical syndrome observed after Taxol treatment in humans.

Cliffer KD, Siuciak JA, Carson SR, Radley HE, Park JS, Lewis DR, Zlotchenko E, Nguyen T, Garcia K, Tonra JR, Stambler N, Cedarbaum JM, Bodine SC, Lindsay RM, DiStefano PS. Physiological characterization of

Taxol-induced large-fiber sensory neuropathy in the rat. Ann Neurol 1998;43:46-55

Neuropathy is a dose-related toxicity of several cancer chemotherapeutic agents such as vincristine, cisplatin, and Taxol. Taxol (paclitaxel) is an effective chemother- apeutic agent that is becoming widely how- ever, it produces a predominantly sensory neuropathy when administered at single doses approaching 250 mg/m2 or at lower doses with repeated The incidence of Taxol-induced neuropathy is often a lot more than 50% with particular dosing regimes, and can reach more than 90%.l Susceptibility to neu- ropathy with Taxol administration is particularly high in populations with other predispositions to neuropa- thy, such as previous chemotherapy, alcoholism, or di- abetes. Early symptoms of Taxol-induced neuropa- thy include numbness and tingling in the distal e ~ t r e m i t i e s . ~ ~ ~ Neurological examination commonly reveals elevation of vibratory thresholds and loss of deep tendon reflexes, consistent with large-fiber sensory dysf~nction.'-~ Electrophysiologically, sensory conduc- tion velocities and amplitudes of sensory nerve action

potentials are reduced and H reflexes are absent or prolonged in l a t e n ~ y . ~ ~ ~ ~ ~ , ~ Histopathologically, severe Taxol-induced neuropathy is characterized by nerve fi- ber loss, axonal atrophy, and demyelination in the sural nerve. Small-fiber sensory modalities or motor systems are sometimes affected, although this is less frequently ~ b s e r v e d . ~ , * , ~ , ~

An effective treatment for chemotherapy-induced neuropathy has not been developed. To assess such treatments, appropriate animals models are important. Taxol-induced neuropathy has been reported after in- traperitoneal injections in rats and mice, with various effects reflecting peripheral sensory neuropathy, includ- ing reduced amplitudes" or conduction velocities' ',12

in peripheral nerve recordings, and anatomical changes in the peripheral nerve." A limitation of these studies is a detrimental effect of Taxol on general health. l 1 In addition, use of growing animals",12 raises the ques- tion of whether the observed effects are due to inter-

8

From Regeneron Pharmaceuticals, Inc, Tarrytown, NY.

*Present address: Millenium Pharaceuticals, Cambridge, MA.

Received Jun 17, 1997, and in revised form Aug 28. Accepted for publication Sep 2, 1997.

Address correspondence to Dr Cliffer, Regeneron Pharmaceuticals, Inc, 777 Old Saw Mill River Road, Tarrytown, NY 10591.

46 Copyright 0 1998 by the American Neurological Association

ference with development o r due to creation of a frank neuropathy i n mature nerve.

In the present study we have developed a model of Taxol-induced neuropathy that preserves the general health of mature animals. W e have used a battery of repeated electrophysiological recordings, histology, and behavioral tests t o provide a comprehensive character- ization of experimental toxic peripheral neuropathy in- duced by Taxol. T h e results support the applicability of this model to the primarily sensory peripheral neurop- arhy that is observed in humans after Taxol treatment.

Materials and Methods Animals, Taxol, and Injections All animal use in this study was conducted in compliance with approved institutional animal care and use protocols and according to N I H guidelines (Guidefor the Care and Use of Laboratory Animals, NIH publication no. 86-23, 1985). Female Sprague-Dawley rats weighing 320 to 450 g were injected intravenously according to one of the following three injection schedules: (1) In the daily schedule, injections of Taxol (3-7 mg/kg) were made in the tail vein of re- strained animals once daily for 5 consecutive days; (2) in the weekly schedule, injections (6-18 mg/kg) were made once weekly in the jugular vein (exposed through a small incision, alternating sides) of anesthetized animals (ketamine/xylazine, 50/10 mglkg ip), for 2 to 6 weeks; and (3) in the twice- weekly injection schedule, a total of two injections (12-18 mg/kg) were made in the jugular veins of anesthetized ani- mals 3 days apart.

Taxol was obtained either as an injectable solution of 12 mg/ml in Cremophor/ethanol (50:50, vol/vol) (gift of Bristol-Myers Squibb, Syracuse, NY), or as a powder (RBI, Natick, MA) that was dissolved in Cremophor/ethanol to a concentration of up to 15 mg/ml. Solutions in Cremophor/ ethanol were diluted 1:i with sterile 0.9% sodium chloride for injection. Thus, for a dose of 15 mg/kg, a rat weighing 400 g was injected with 1.0 ml of a solution of 6 mgfml Taxol.

Electrophysiological Tests For electrophysiological recordings, animals were anesthe- tized by using ketaminelxylazine (50-100 mg/kg and 5-10 rng/kg, respectively) intraperitoneally, supplemented as needed. Rectal temperature was maintained between 37 and 38.5"C by using a feedback-controlled heating pad. Foot or tail temperature was kept stable at 35 -C 0.3"C by using a feedback-controlled heating lamp connected to a small ther- mocouple (IT-1 8; Physitemp, Clifton, NJ) inserted subcuta- neously near the recording site.

Hindlimb Recording M AND H WAVES. The paradigm for repeated hindlimb stimulation and recording was a variation of that used pre- viously to describe severe H-wave amplitude deficits in a modcl of pyridoxine-induced large-fiber sensory neuropathy, with associated behavioral and anatomical abn0rma1ities.I~ A

schematic diagram of the recording setup is shown in Figure 1. Stainless steel stimulating cathodes were inserted at the ankle (Achilles tendon) and hip (sciatic notch), near the tib- ial and the sciatic nerves, respectively. Anodes were 1 cm proximal to the cathodes. A reference recording electrode was inserted subcutaneously into the fifth digit of the hindlimb and a ground electrode was placed at the base of the tail. The location on the foot for the recording electrode was determined during supramaximal stimulation at the ankle. A stainless steel electrode was hooked subcutaneously on the dorsum of the foot at a location where a maximal M wave could be recorded while maintaining amplitude of the H wave.

Square-wave stimuli of constant current and 0.2-msec du- ration were delivered at a frequency of 0.2 to 0.3/sec. Re- corded output was amplified lOOX and sent to a storage oscilloscope and to a computer-based data-acquisition system (Cambridge Electronic Design, Cambridge, UK) at 20,000 samples/sec for later analyses. Amplifier high-frequency cut- off filters were set for 3, 5, or 10 kHz.

Stimulating-cathode positions were adjusted to achieve thresholds for the M or H wave of 0.5 mA or less. Measure- ments were made on averaged responses to eight stimuli. Re- cordings were obtained during stimulation of the tibial and sciatic sites at an intensity to maximize the H-wave ampli- tude (HmJ and at 1.2X the minimum stimulus adequate to maximize the M-wave amplitude The distance be- tween the tibial and sciatic cathodes was divided by the dif- ferences in M- and H-wave latencies from the two locations (to the first major negative peaks) to calculate motor and sensory conduction velocities. Peak-to-peak amplitudes were measured for the M and the H waves, and the Hm,/Mm,x ratio was calculated to provide a measurement of the H-wave amplitude normalized to the M-wave amplitude.

Tail Recordings Repeated tail stimulation and recording were done through bare 26-gauge stainless steel needle electrodes (Jari Electrode, Yucca Valley, CA) inserted subcutaneously on the side of the tail. The arrangement, a variation of those used previously for caudal nerve is shown schematically in Fig 1. General stimulation and recording parameters were the same as for the leg. Stimuli were administered at a fre- quency of l/sec. Three pairs of stimulating/recording elec- trodes were inserted (referred to as A, B, and C; see Fig 1). A ground electrode was inserted between the stimulating and recording pairs of electrodes. Supramaximal stimuli (1.2 X the minimal stimulus adequate to maximize the response).

M WAVES. Electromyographic (EMG) recordings from segmental tail muscles were made by stimulating through a proximal pair of electrodes (distal electrode as cathode) and recording the bipolar potential from a more distal pair (see Fig 1). An interelectrode distance of 10 mm provided an isolated recording of the muscles from one segment (seg- ments were 10-12 mm long). Electrode pair A was placed near the base of the tail (30-40 mm from the caudal limit of the ischium). The location of the proximal electrode of pair B was determined by using unipolar recordings (refer- ence electrode at the tip of the tail) during supramaximal

Cliffer et al: Taxol Neuropathy in the Rat 47

ELECTRODE PLACEMENT

HIND LIMB RECORDING

SPINAL CORD

'0 H WAVE= SENSORY- I _ - - - - _ _ - _ - _ - - - _ _ _ - -

MEDIATED MUSCLE

TAIL RECORDING - EMGA~OTOR

NERVE -s MUSCLE

Fig I . Schematic drawing of electrophysiological recordings. (Top) Electrode placements. For hindlimb recording in the foot, stimuli were administered at the hip (sciatic site) and ankle (tibial site). The stimulating cathode is designated by a solid line. For tail recording, electrode pairs were placed at three sites-A, B, and C. Each pair was set up to be wed for either stimulation or recording during stimulation, the cathode was toward the pair used for recording. (Middle) Stimulation of sciatic or tibial nerve send action potentials in two directions. Motor jbers activate the muscles in the foot, resulting in an M wave recorded in the foot as an electrornyogram (EMG). Sensory fibers, with their cell bodies in the dorsal root ganglion (DRG), activate motor neurons (MN) in the spinal cord through the monosynaptic reJex pathway, resulting in an H wave, which appears later than the M wave. Stimulation at the hip and the ankle allows calculation of conduction velocities (CVS), where CV = the distance between stimulation sites divided by laten9 differences. (Bottom) For tail recording, stimulation proximally elicits segmental muscle activity more distally, resulting in a mo- tor potential. Stimulation distally allows recording o f a com- pound nerve action potential (CNAP) more proximally. As in the leg, stimulation or recording at more than one site allows calculation of CVS fiom distance and latency differences.

stimulation at A to find a peak EMG amplitude along the tail about 30 mm from A. Electrode pair A was then ad- justed to a location 30 mm proximal to pair B. Pair C was placed by the same method to maximize the recording about 30 to 35 mm distal to pair B.

Measurements were made from traces averaged from eight responses amplified 1OOX. Motor recordings were made at sites B and C during supramaximal stimulation at A, and at site C during supramaximal stimulation at B. Differences in stimulation-recording distances were divided by the corre- sponding latency differences (measured to the initial negative peaks) to obtain motor conduction velocities. Amplitudes were measured from the baseline to the largest negative potential.

COMPOUND NERVE ACTION POTENTIALS. Caudal nerve recordings were used to measure sensory function in the tail; the caudal nerve is composed predominantly of sensory fibers (-90%16). Nerve recordings in the tail were made by stimu- lating through a distal pair of electrodes (proximal electrode as cathode) and recording from a more proximal pair. Because the nerve is not segmentally organized like the muscles, the nerve recordings did not show periodic variability in ampli- tude along the tail. Recordings were made at sites B and A during supramaximal stimulation at C, and at site A during supramaximal stimulation at B. Measurements were made on traces averaged from 32 responses amplified 1,OOOX. Conduc-

tion velocity was calculated as described above for the motor responses, and peak-to-peak amplitudes were measured.

Behavioral Tests Behavioral tests of motor function, sensorimotor coordina- tion, and nociception were performed. Forelimb and hind- limb grip strengths were measured" as tests of motor func- tion. The animal was held by the tail, maneuvered to grasp a metal grid attached to a mounted strain gauge (Model DPP- 10; Chatillon, Greensboro, NC), and pulled back until it released its grip. The average of the maximum forces exerted in three trials was divided by the body weight to determine a weight-normalized grip strength (kg/kg body weight). A test of beam-balancing ability provided measures of sensorimotor coordination requiring proprioceptive function. For each trial, animals were placed on a 4-cm beam, parallel to its axis. Ability to balance was scored on a scale from 1 to 5, where 1 = balancing with steady posture and 5 = falling off with no attempts at balancing." Time (duration) on the beam was measured, with a cutoff of 60 seconds. Median scores and times from three trials were analyzed. Heat noci- ception was tested by using the tail-flick teal9 and the hind- limb withdrawal test,20 using devices from Columbus Instru- ments (Columbus, OH) and UCSD Anesthesia Research and Development (San Diego, CA), respectively. Mean latencies to withdrawal of the tail or foot from a noxious heat source were calculated from three or four trials.

48 Annals of Neurology Vol 43 N o 1 January 1998

Histo Lou Rats were anesthetized and perfused through the aorta with heparinized saline followed by 3% glutaraldehyde in 0.1 M phosphate buffer. Lumbar fourth or fifth dorsal and ventral roots were dissected out, postfixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer in 10% sucrose (pH 7.4), embedded in plastic, cut at 0.5 pm, mounted on slides, and stained with 1% toluidine blue in 1% borate buffer.

Statistical Tests Repeated-measures analyses of variance were used for para- metric measurements (Statview, Abacus Concepts, Berkeley, CA). Post hoc comparisons among groups were by Fisher's protected least significant difference (PLSD). Single-time comparisons in experiments in which there was an overall effect used Student's t tests with Bonferroni correction for multiple tests. Categorical data for beam performance, and for normal (60 seconds) versus abnormal (<60 seconds) beam durations, were tested for differences between treat- ments in overall distribution over cime, using a Cochran- Mantel-Haenszel test (JMP Statistical Discovery Software, SAS Institute, Cary, NC). If the overall test showed a differ- ence between groups, individual weeks were tested by using x2 analysis. Significance was ascribed to p 5 0.05.

Results Establishment o f the Model To establish a protocol for Taxol dosing that would produce a reproducible neuropathy while maintaining the animals' general health, we tested various schedules of intravenous administration, which is the route used in humans. Comparisons of changes in H,,/M,, ra- tios and in compound nerve action potential ampli- tudes, among selected doses and schedules of Taxol ad- ministration, are provided in Table 1. Values in Table 1 are averages of the maximum differences from vehicle-treated animals during experiments of a given design, selected from a total of 14 experiments (one to five of each design are represented in Table 1).

Comparisons among the Taxol-dosing regimens show clearly that the severity of the neuropathy and the effects on other aspects of animal health depended

on both the dose and the frequency of administration of Taxol, as opposed to solely the cumulative dose ad- ministered over time. Within each schedule, severity of the neuropathy increased with increasing dose (see Ta- ble 1; also see Fig 5). However, the daily-injection schedule resulted in severe weight loss (Fig 2A) and death in 50% of the animals treated with 5 or 7 mg/kg daily, by 2 weeks after treatment. Despite deteriorating health, the group receiving 5 mg/kg did not exhibit a significant neuropathy, whereas those receiving 7 mg/kg did (see Table 1). The weekly schedule resulted in mild weight deficits and no morbidity or mortality, creating a dose-dependent neuropathy with doses of 15 to 18 mg/kg over 4 to 5 weeks. More rapid induction of the neuropathy with preservation of the animals' health resulted from a total of two injections of 12 to 18 mglkg Taxol given 3 days apart, the twice-weekly injection schedule. Only a mild weight deficit devel- oped during the 2 weeks after injection (see Fig 2B), with no morbidity or mortality. The severity of the neuropathy in this paradigm was also dose dependent, measured both by evoked sensory amplitudes (see Ta- ble 1 and Fig 5 ) and by results of the beam-balancing test (not shown). The importance of injection fre- quency in determining the severity of the neuropathy is illustrated by several comparisons. Doses of 15 or 18 mg/kg produced a less severe neuropathy after weekly injections over 4 or 5 weeks than when the same dose was given twice in 3 days, for about half the cumula- tive dose. Similar levels of neuropathy were induced by 7 mg/kg given daily for 5 days, by 15 mg/kg given twice in 3 days, and by 18 mg/kg given weekly for 4 weeks (to a much greater cumulative dose). The twice- weekly injection schedule was adopted for further test- ing, based on its simplicity, rapid induction of neurop- athy, and preservation of general health.

Histopatho Logy The predominantly sensory nature of the neuropathy induced by Taxol treatment is revealed by the histolog-

Table 1. Summary of Pbysiological Results fiom Experintents with Selected Dosing Regimens

H,JMrnax CNAP Amps Schedule Dose (mg/kg) No. of Doses Cumulative Dose (Yo of Control) (% of Control)

Daily 5 5 25 90 72 7 5 35 17" 47"

Weekly 15 5 75 79 63" 18 4 72 16" 35"

18 2 36 5" 26" Twice weekly 15 2 30 20" 39"

H,,IM,, ratios (from recordings using tibia1 stimulation) and the compound nerve action potential (CNAP) amplitudes (Amps) recorded in the tail are presented as percentages of values in vehicle-treated rats (% of Control); thus, lower values indicate more severe neuropathy.

"Significant difference ( p < 0.05) from control (analysis of variance with post hoc comparison, using Fisher's PLSD).

Cliffer et al: Taxol Neuropathy in the Rat 49

A o Vehicle B o Vehicle + Taxol, 7mglkg + Taxol, 18 mglkg

440 -

8 4 0 0 - E m 5 360 -

o 320 -

- ._

>, u

11111 4

i t 280 G

280 +

OA I I I I I I 1 O A , I j I , , , 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Weeks Weeks

Fig 2. Body weights of Taxol-treated rats. (A) Animals receiu- ing five daily doses of 7 mg/kg (arrows) showed a precipitous weight loss, resulting in 50% mortality. The rapid recovery between weeks 1 and 2 is partially due to dropout of the most severely afected animals. (B) Animals receiving two I8-mg/kg doses on the twice-weekly schedule (arrows) showed a mild weight loss over week I and then recovered. Coeficients o f variabiliq rangedfrom 4 to IG%.

ical results. In animals receiving two doses (twice weekly) of 15 mg/kg, examination of the L4 dorsal root collected 4 weeks after Taxol administration re- vealed reductions in axon caliber and density and de- generated myelin profiles (Fig 3). Dark axonal profiles indicating Wallerian degeneration were apparent. The presence of small dark profiles suggested aborted at- tempts at regeneration. Ventral roots of Taxol-treated rats appeared normal. Because Schwann cells myelinate both motor and sensory fibers, the results suggest that the severe pathology we observed is due to sensory neu- ronal or axonal damage rather than a primary lesion of Schwann cells.

Electrop hysio lo^ The most robust electrophysiological changes observed after Taxol treatment were the deficits in evoked sen- sory amplitudes. Figure 4 shows traces from control and Taxol-treated animals. Amplitudes of H reflexes in the hindlimb were diminished severely. In contrast, motor amplitudes were unaffected. Corresponding changes were observed in the tail recordings; the com-

Fig 3. Photomicrographs of dorsal roots and ventral roots from animals treated with vehicle or 15 mg/kg Taol in the twice-weekly injection paradigm. Roots were sampled 4 weeks aJer treatment. Dorsal roots from Taxol-treated animals showed severely reduced density of intact myelinated fibers, numerous fibers undergoing Wallerian degeneration, and many small dark profiles sugesting aborted attempts at regeneration. Ventral roots from Taxol-treated rats appeared normal. Scale bar = 100 Fm.

50 Annals of Neurology Vol 43 No 1 January 1998

A HIND LIMB

Tibia1 stimulus Sciatic stimulus

2msec M

B TAIL EMG (motor) CNAP (sensory)

2 msec 2 msec

Fig 4. Electrophysiological recordings from the hindlim b (A) and the tail (B) in vehicle-treated and Taxol-treated rats. (A and B) Top traces: Vehicle treatment. Bottom traces: Two twice-weekly injections o f 18 mglkg Taxol. (A) Hindlimb re- cordings were in response to stimulation (arrows) of the tibial (lej) or sciatic (right) nerve. Traces were recorded with supra- maximal stimulation for the M waves. (B) Tail recordings over the proximal portion of the tail are illustrated The traces at the lefi (EMG) are electromyograms recorded from site B (see Fig 1) during stimulation at site A; the traces at the right (CNAP) are compound nerve action potentiah recorded from site A during stimulation at site B. Calibrations are shown for each type of recording. Stimulus art$& in the nerve record- ings have been truncated for clarity.

pound nerve action potentials were diminished, but motor amplitudes were maintained. Vehicle injections alone did not produce differences in amplitudes ot conduction velocities from those recorded in saline- injected control animals. Figure 5 presents a summary of the changes observed in Taxol-treated animals, over the I0 weeks after treatment in one experiment, in which two doses ( I2 and 15 mg/kg) were administered in the twice-weekly dosing paradigm. The reduction in H-wave amplitude was evident by 1 week after treat- ment (see Fig 5C), whereas the reduction in tail-nerve amplitude took 2 weeks to develop (see Fig 5E). These amplitude reductions showed no significant recovery, even 16 weeks after twice-weekly treatment with 15 mg/kg (data not shown). In contrast, 18 weeks after induction of neuropathy with the daily-injection para- digm (7 mg/kg), H waves returned to control levels in

HIND LIMB A M Amplitude (mV) 30 1 ++-

0 4 8 1 2

c H,,JM,,, ratio

o.6 1 I+

0.4 1 f T

0 4 8 1 2

TAIL NERVE E Amplitude (pV)

0 4 8 1 2 Weeks

B Motor CV (mlsec)

6o 1 ++

30 0 , 4 , I I 0 4 8 1 2

D Sensory ~ ~ ( m / s e c )

F CV (mlsec)

Vehicle 6 Taxol, 12 mglkg

30 v Taxol, 15 mglkg

0 4 8 1 2 Weeks

Fig 5. Dose-response and time course of electrophysiological parameters measured during an experiment using the twice- weekly Taxol-injection paradigm (12 and 15 mg/kgl Times of Taxol injection are indicated by arrows. Recordings were made 1, 2, 5, 8, and 10 weeks after the second Taxol injection. Effects on H,JM,, ratios and tail-nerve amplitudes were dose dependent. Conduction velocity (Cv) deficits were modest compared with those of amplitudes, consistent with a primarily rwonal, rather than demyelinating, neuropatby. Asterisks indi- cate significant overall differences in this experiment between Taxol-treated groups and the vehicle-treated control group (repeated-measures analysis of variance; Fisher i PLSD for comparisons among groups; p < 0.05; ‘9 5 0.005).

surviving animals, whereas tail-nerve amplitudes re- mained reduced (data not shown).

Modest deficits were observed in both motor and sensory conduction velocities. In the hindlimb, these changes developed during the 2 weeks after treatment, and recovered to control levels by 10 weeks for the I2 mg/kg dose (see Fig 5B and D). Motor conduction ve- locity recovered by 16 weeks for the 15 mg/kg dose (data not shown). Nerve conduction velocity in the tail was also reduced by Taxol (see Fig 5F; significant for the 15 mg/kg dose in combined experiments). Motor conduction velocity in the tail was reduced signifi- cantly, for the twice-weekly 15 mg/kg dose of Taxol,

Cliffer et al: Taxol Neuropathy in the Rat 51

only 8 weeks after Taxol treatment (data not shown). Tail-nerve conduction velocity recovered to control lev- els by 8 weeks for the 12 mg/kg dose (see Fig 5F) and by 16 weeks for the 15 mg/kg dose (data not shown).

Latencies of H and M waves in the hindlimb showed increases with Taxol treatment consistent with the decreased conduction velocity in both the sensory and the motor portions of the reflex pathway. Taxol- treated animals were not larger in size, and stimulation- recording distances were comparable in the two groups, indicating that the differences in latency were not due to differences in conduction distance. The latency of the H wave from both tibial and sciatic stimulation increased significantly (Table 2; see Fig 4) . The path- way for evoking the H wave includes a central synapse and the motor pathway (see Fig 1). Therefore, the H-wave latency increase could be partially due to an increase in the central synaptic delay as well as to re- ductions in both sensory and motor conduction veloc- ities. The increased M-wave latency, reflecting the de- crease in motor conduction velocity, was significant only for sciatic stimulation (see Table 2), which pro- vides a longer pathway over which to discriminate a change than does tibial stimulation.

Behavior The performance of Taxol-treated rats in the behav- ioral tasks was consistent with the histopathological and electrophysiological changes showing deficits pref- erentially in large sensory fiber structure and function. The time course of changes in beam performance is shown in Figure 6 for animals given a twice-weekly dose of 15 mg/kg Taxol. Deficits in sensorimotor co- ordination were reflected in the increased performance scores and reduced time on the beam. Beam scores

Table 2. Latencies for a Group o f Animals 4 Weeks after Tmol Treatment"

A B

0 2 4 6 8 1 0 0 2 4 6 8 1 0 Weeks Weeks

Fig 6 Changes in sensorimotor performance in rats treated twice weekly with 15 mg/kg Tax01 (arrows). (A) Median beam score. (B) Beam duration; mean % SEM. Asterisks in- dicate signiJicant differences versus vehicle-treated control p o u p (2; 'r, < 0.05; *p < 0.01).

were abnormal from the first through fifth weeks after treatment (see Fig 6A). Scores recovered to baseline levels between weeks 5 and 10. In additional experi- ments with different testing schedules, beam scores re- covered to control levels between weeks 6 and 8 after Taxol treatment (data not shown). Beam duration was reduced 1 week after injections and remained low for several weeks thereafter; as with beam scores, these val- ues also recovered by 10 weeks (see Fig 6B). In animals dosed twice (twice weekly) with 12 mg/kg, beam scores and beam durations were not significantly different from controls (not shown). This contrasts sharply with the significant electrophysiological deficits observed in this group (see Fig 5).

Neither the grip-strength tests to assess motor func- tion nor the heat nociceptive tests for small-fiber sen- sory function revealed deficits in Taxol-treated animals compared with vehicle-treated animals. The absence of deficits in grip strength was consistent with both the lack of motor amplitude changes and the lack of ven- tral root pathology.

Latenciesb Discussion (msec) Vehicle (n = 5) Taxol (n = 8) The results of the present study show that Taxol ad- M wave

Tibial 1.42 i 0.06 (0.03) 1.44 -+ 0.10 (0.04)

Sciatic 2.90 2 0.12 (0.06) 3.12 2 0.12 (0.04)' stimulation

stimulation H wave

Tibial 7.75 -f 0.14 (0.06) 8.42 i 0.58 (0.20)d

Sciatic 6.34 -+ 0.15 (0.07) 6.88 i 0.43 (0.15)d stimulation

stimulation

"Treatment was two doses of 15 mg/kg on the twice-weekly schedule. bLatencies were measured to first deflection of the major wave from the baseline. Data are mean 2 SD (SEM). Significant difference conipared with vehicle-treated animals: 'p < 0.01; 'p < 0.05 (Student's t test).

ministered intravenously twice in 3 days to mature rats produces a robust, reproducible large-fiber sensory neu- ropathy without appreciable adverse effects on weight and general health. This experimental neuropathy par- allels the Taxol-induced neuropathy observed in hu- mans. Although reduction in both sensory and motor conduction velocities in the periphery suggests involve- ment of peripheral Schwann cells, the most severe pa- thology was in large sensory fibers. This pathology was evidenced by (1) the specific lesion in dorsal roots, (2) the diminished sensory-mediated amplitudes in electro- physiological recordings, and (3 ) the deficits in a be- havioral task requiring proprioceptive function. The se- verity of the pathology in the dorsal roots under the

52 Annals of Neurology Vol 43 No 1 January 1998

administered dose indicates a neuronopathy rather than a strictly peripheral, length-dependent axonopathy. A milder dosing regimen might produce a more selec- tively peripheral axonopathy. It is noteworthy that in- duction and severity of the neuropathy in the rats de- pends on both dose and frequency of administration of Taxol. This dependence corresponds to the observation that Taxol-induced neuropathy in humans with a set dosing frequency depends not only on the cumulative dose, but also on the size of the individual dose ad- ministered.2 An important aspect of the twice-weekly dosing paradigm described here is that it lends itself well to titration of the dose to produce any level of neuropathy, from mild to severe. Thus, this model af- fords many advantages for studying potential ameliora- tive therapies for Taxol-induced neuropathy.

The most salient advantage of this model is the re- production of the predominantly large-fiber sensory neuropathy that is induced by Taxol treatment in hu- mans. The reported incidence of neuropathy in various patient populations with different Taxol-dosing regi- mens ranges from less than 20% to more than 90%, with most reports describing a predominantly sensory neuropathy.’ Patients treated with sufficient doses of Taxol experience numbness and tingling in the dis- tal extremities, often within days after a single large d ~ s e . ~ , ~ Neurological examination of affected patients commonly reveals loss of large fiber-mediated sensory abilities, namely, vibration and proprioception, as well as diminished or absent deep tendon r e f l e ~ e s . ~ - ~ * ~ , ~ ~ Electrophysiological examination of affected humans shows a corresponding reduction in sensory nerve ac- tion potential amplitude or conduction velocity and abolition or prolonged latencies of H r e f l e ~ e s . ~ ~ ~ ” . ~ The results of our electrophysiological recordings in the tail nerve and hindlimb in the rat model parallel these clinical findings directly. Histopathological descriptions of Taxol neuropathy in humans are limited; sural nerve biopsy of a severely affected patient revealed reduction in myelinated fiber density and substantial axond atro- phy.’ We made corresponding observations on the dorsal roots of Taxol-treated animals.

Muscle weakness is not commonly observed after Taxol treatment in humans.’ Only after repeated, pro- longed Taxol treatment were motor conduction abnor- malities detected in patient^.^ In those patients treated with both Taxol and cisplatin, motor amplitude defi- cits were more common than in patients treated with Taxol alone.2 In our rat model, we observed deficits in motor conduction velocity, but not in amplitude, in- dicating mild motor involvement accompanying the sensory deficits. Furthermore, neither anatomical nor behavioral evidence indicated substantial motor abnor- malities in the rats.

Like motor involvement, the incidence of small-fiber sensory abnormalities in Taxol-treated patients is in-

f r e q ~ e n t . ~ , ~ Small-fiber sensory symptoms, when ob- served, include loss of pinprick or temperature sensa- t i ~ n . ~ - ‘ We did not detect abnormalities in the small fiber-mediated thermal nociceptive responses in the rat model at the dose of Taxol used (15 mg/kg, twice weekly). However, deficits in small-fiber sensory func- tion or in motor function might be produced, as in humans, by higher doses or more prolonged treatment with Taxol.

Aside from reliably reproducing important features of the expression of the neuropathy in humans, our model offers additional advantages over other models of chemotherapeutically induced toxic peripheral neu- ropathies in two important ways. First, systemic toxic- ity that compromises animal health can make difficult the induction of neuropathy before animals become unacceptably ill, and can confound interpretation of a neuropathy that does develop. For instance, cisplatin causes not only neuropathy, but substantial nephropa- thy.22223 Nephropathy can, in turn, induce an associ- ated uremic n e ~ r o p a t h y . ~ ~ Thus, the neuropathy in cisplatin-treated animals may be secondary to ure- mia instead of or in addition to being directly caused by the cisplatin. In addition, body weight loss and cachexia may have effects on nutritive or trophic influences on nerve function. Adverse effects of Taxol treatments on the health and activity of the animals have been previously noted after intraperitoneal injec- tions. In our experiments, animals given Taxol intravenously twice weekly (or weekly) did not show generalized illness or activity changes. Weight loss was minimal and recovery was rapid, similar to what occurs in humans.26

A second important advantage of the model we have presented here is the use of mature female rats of stable size and body weight, rather than animals undergoing developmental or maturational changes. Studies using conduction velocities as measures of peripheral nerve function often report an increase in conduction veloc- ity in the control population, which explains much or all of the eventual difference from the experimental p~pu la t ion .~ ’ -~~ Such patterns have been observed in studies of Taxol’s effects on electrophysiological param- eters. In such situations, it is not clear whether the neuropathy can represent compromise of function in a fully developed nerve or only interference with devel- opment in a growing nerve.

Despite differences in design of previous Taxol mod- els from that reported here, comparisons of results re- veal both similarities and differences. Reduced sensory- related conduction velocity was reported in peripheral nerves in rats treated intraperitoneally with Taxol,’ ‘,12

which corresponds to our findings for the caudal nerve and for fibers mediating the H reflex. Apfel and col- leagues” reported reduced amplitude in the caudal nerve of mice given Taxol intraperitoneally, similar to

11,12

Cliffer et al: Taxol Neuropathy in the Rat 53

our results in rats treated intravenously. However, these same authors found no significant effect of Taxol on latency of caudal nerve recordings. In addition, Con- treras and associatesA5 reported no change in velocity or amplitude in recordings from several nerves in the same model, and Campana and colleagues25 found changes in neither motor nor sensory peripheral nerve conduction velociry in rats treated intraperitoneally with substantial cumulative doses of Taxol. In contrast, we did find reductions in sensory and motor conduc- tion velocity and in sensory amplitude in recordings both from the caudal nerve and from the hindlimb. Altered nociceptive responses to heat stimuli were pre- viously reported in and treated intra- peritoneally with Taxol, whereas changes in heat noci- ceptive responses were not significant in our experiments. Anatomically, Cavaletti and co-workers’ reported aggregates of neurotubules, edema, and high numbers of small myelinated fibers in peripheral nerves after Taxol treatment in rats. However, compared with our observations, the anatomical changes reported by these authors in the dorsal roots were mild, including neither fiber degeneration nor abnormalities of myeli- nation. The difference may be due to a milder dosing regimen (16 mg/kg weekly, comparable with our 15 mg/kg weekly, which did not produce neuropathy; see Table 1). However, when Taxol was injected directly into nerves, local degenerative and regenerative reac- tions were ~ u b s t a n t i a l , ~ ~ similar to our observations in the dorsal root after systemic delivery.

In the weeks after Taxol administration in the present study, behavioral manifestations of the neurop- athy developed rapidly, then diminished, while the electrophysiological evidence of peripheral damage re- mained substantial. In addition, we observed electro- physiological deficits at a dose not showing robust behavioral deficits (12 mg/kg). These observations in- dicate that behavioral abilities could be maintained in the face of a low level of peripheral damage, and could recover despite continued peripheral damage. Maintenance of sensory-motor coordination in the face of electrophysiological evidence of neurological damage has been observed for pyridoxine-induced neu- ropathy. l 3 Maintenance or recovery of behavioral abil- ities may be mediated by plasticity in central connec- tions of the affected peripheral fiber populations, as well as by compensation using alternative behavioral strategies involving intact circ~its.~’

Electrophysiological testing of animal models of pe- ripheral neuropathy often focuses on assessment of conduction velocities or latencies without measurement of amplitudes.’ 1,12,28338 A comprehensive assessment of neuropathy in animal models can be achieved by com- bining measures of sensory and motor amplitude and conduction velocity with evaluation of behavior and anatomy. The prominence of sensory amplitude reduc-

tions, over the more mild conduction velocity changes due to Taxol, indicates that loss of functioning sensory fibers is a more important contributor to the patho- physiology of Taxol-induced neuropathy than demyeli- nation. In the Taxol model described here, multiple techniques have been exploited to demonstrate an ex- perimental neuropathy that closely resembles the neu- ropathy seen in Taxol-treated patients. Additional ad- vantages of this model, over other available models of chemotherapeutically induced toxic neuropathy, in- clude the simple dosing regimen, rapid induction, abil- ity to vary severity of the neuropathy, use of mature animals, and absence of substantial systemic toxicity. These features make this model ideal to test agents for the treatment of Taxol-induced neuropathy.

We thank Drs Andrew Mizisin, Maureen Helgren, and Philip Low and Mr Jim Schrnelzer for helpful discussions and assistance, Dr Robert Buroker and Bristol-Myers Squibb for providing Taxol, and Claudia Murphy for assistance with graphics.

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