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The effect of chemotherapeutic agents upon peripheral nerves
The Effect of Che.totherapeutic Agents upon Peripheral Nerves BARNES WOODHALL, M.D., S T E P H E N MAHALEY, JR., M . D., S T E P H E N A N D H A R R Y H U N E Y C U T T , M . D . , Ouke University Medical School
The first indication that specific changes might occur in peripheral nerve tissue following exposure to c e r t a i n a n t i t u m o r a g e n t s d e v e l o p e d f r o m the o b s e r v a t i o n t h a t r e l i e f of p a i n f r o m t h e t u m o r a r e a o c curred after regional chemotherapeutic perf u s i o n , ls'm'2''22 P a t i e n t s t r e a t e d w i t h l a r g e intravenous dosages ofnitrogenmustard, with s p e c i a l t e c h n i q u e s f o r the p r o t e c t i o n of b o n e marrow, also developed focal cranial nerve d e f i c i t s i n the f o r m of h e a r i n g l o s s . z. t0,23 S u b s e q u e n t l y , n i t r o g e n m u s t a r d - - C 14 i n j e c t e d i n t r a v e n o u s l y i n d o g s w a s f o u n d to l o c a l i z e s e l e c t i v e l y in the eighth n e r v e , a m o n g other c r a n i a l n e u r a l s t r u c t u r e s . Iz P e r i p h e r a l n e u r o p a t h i e s of the l o w e r e x t r e m i t i e s h a v e b e e n r e p o r t e d a s a s e q u e l of r e g i o n a l p e l v i c p e r fusion with mustards3"17 and intravenous therapy with v i n c a l e u k o b l a s t i n e , s. 19.20 T h e s e d o c u m e n t e d e v i d e n c e s of n e u r o t o x i c drug a c t i o n in the p e r i p h e r a l and c r a n i a l nerve systems, together with earlier evidence c o n c e r n e d with the c e n t r a l n e r v o u s s y s t e m , s s t i m u l a t e d t h i s s t u d y of the e f f e c t of c h e m o t h e r a p e u t i c a g e n t s u p o n t h e s c i a t i c n e r v e of the dog. MATERIALS AND METHODS The drugs used in these experiments were: methyl-bis (fl-chloroethyl)-amine hydrochlor i d e (HN2); 2 , 5 - d i e t h y l e n i m x n o - 3 , 6 - b i s ( 2 - m e t h o x y e t h o x y ) - 1 , 4 - b e n z o q u i n o n e (A139); v i n c a l e u k o b l a s t i n e s u l f a t e ( V L B ) ; 2 H - l , 3, 2oxazaphosphorine, 2-bis(chloroethy1) amino-
F r o m the Division of N e u r o s u r g e r y , the Duke U n i v e r s i t y Medical School, Durham, North Ca rolina. Supported by U.S.P.H.S. Grants # M 2109, H 3582 and CY 4751, from the National Institutes of Health, Bethesda, Maryland. Submitted for publication December 26, 1961. ]$R -
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BOONE,
M . D.,
tetrahydro-2-oxide (Cytoxan); and 6-purine f l - D - g l u c o t h i o p y r a n o s i d e (6-1viPG). L e s s i n t e n s i v e s t u d i e s w e r e c a r r i e d out with p - b i s (2-chloroethyl) a m i n o p h e n y l a l a n i n e (PAM), Actinomycin D and Methotrexate. Each drug was dissolved in Ringer's solution (except PAM, which was dissolved in propylene glycol). Each sciatic nerve was exposed in the t h i g h b y s e p a r a t i n g t h e b i c e p s f e m o r i s and the s e m i t e n d i n o s u s m u s c l e s . I n t h e f i r s t g r o u p of e x p e r i m e n t s a f e l t p l e d g e t 1 c m . s q u a r e w a s p l a c e d u p o n the epineurium. The pledget was saturated with 0.1 c c . of the d r u g s o l u t i o n ( e x p e r i m e n t a l ) or R i n g e r ' s s o l u t i o n (control). A f t e r five m i n u t e s t h e p l e d g e t w a s r e m o v e d a n d the i n c i s i o n w a s c l o s e d . I n t h e s e c o n d g r o u p the d r u g o r d i l u e n t (0.1 c c . ) w a s i n j e c t e d b e n e a t h the e p i n e u r i u m . I n t h e t h i r d g r o u p a l o w e r l i m b w a s r e g i o n a l l y p e r f u s e d f o r 20 m i n u t e s v i a the f e m o r a l a r t e r y a n d v e i n . T h e d r u g w a s m i x e d u n i f o r m l y i n t h e p e r f u s a t e , 500 c c . of w h o l e b l o o d a t 37 ~ C. T h e e x t r a c o r poreal circuit consisted of a m o d i f i e d Travenol bubble oxygenator, a Harrison heat exchanger, a bubble trap, a rotameter flowm e t e r and a S i g m a m o t o r pump. P a r a m e t e r s m e a s u r e d w e r e systemic arterial pressure, perfusion arterial pressure, perfusate temperature and perfusion flow. Following perfusion the vessels w e r e repaired withS-0 arterial silk. A control perfusion was done in one dog with no chemotherapeutic agent in the perfusate. Five days after drug administration, each sciatic nerve w a s taken for h i s t o l o g i c e x a m i n a t i o n ; i n s o m e c a s e s 20 d a y s w e r e a l l o w e d to e l a p s e b e t w e e n n e r v e injection and histologic examination. Cross and longitudinal sections were stained with hematoxylin and eosin, Bodian's solution, luxol-fast-blue and periodic acid--Schiff stains. Nerve conduction studies w e r e p e r f o r m e d across the site of subepineurial injection of 373
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WOODHALL, MAHALEY, BOONE a n d [ [ U N E Y C U T T
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Drug
Histologic Nerve Changes
0.001 0.005
Demyelinization~ thickening of epineurium Demyelinization~,. thickening of eplneurium, migration of axis cylinders laterally DemyellnizationI thickening of eplneurium, localized intrafascicular hemorrhage and edema; endoneurlal thickening; axis ay[{nder swe[llng and disappearance Same as above, with more diffuse changes and mare marked endoneurial thicken{ng Same as above
HN2 0.1 1.0 A139
0.001 0.01 0.1 0.0001 0.001 0.01
VLB 0.1
The histologic changes noted following drug administration bypledget, subepineurial injection and perfusion are listed in Tables i , Z a n d 3, r e s p e c t i v e l y (the h i s t o l o g i c c h a r a c t e r i s t i c s of n o r m a l p e r i p h e r a l n e r v e s h a v e been described It). The smallest concentrat i o n of HN2 a d m i n i s t e r e d by pledger or subepineurial injection caused swelling, pale s t a i n i n g a n d d i s o r g a n i z a t i o n of t h e m y e l i n , a s w e l l a s a f i b r o b l a s t i c r e s p o n s e a l o n g the
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Dose (mg./kg.)
0.01
RESULTS
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November,
Table 1, Histologlo Changes in the Sciatic Nerve of the Dog Five Days Following Five-Minute Pledget Application of Chemotherapeutic Agents*
drug or Ringer's solution. Abipolarplatinum stimulating electrode was placed onthe nerve 6 c m . p r o x i m a l to t h e i n j e c t i o n s i t e , a n d b i p o l a r p l a t i n u m r e c o r d i n g e l e c t r o d e s (5 m m . a p a r t ) w e r e p l a c e d 2 c m . d i s t a l to the i n j e c t i o n 9 s i t e . T h e n e r v e w a s s t i m u l a t e d (3 v o l t s ) w i t h a G r a s s s t i m u l a t o r ( s i n g l e , m o n o p h a s i c 0.01 msec. impulses) and isolation unit, andnerve potentials were recorded distally by a Grass P - 6 p r o b e a n d D . C . a m p l i f i e r , fed i n t o a DuMont oscilloscope and Grass Kymograph camera. Records were taken before nerve injection, immediately after injection and five days later. Drug dosages that produced histologic nerve changes were chosen for conduction studies.
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1.0
Cytoxan
6-MPG
Demyelinization; intrafascicular edema Same as above, but more marked Same as above, plus axis cylinder flagmentation Normal Normal Localized demyelJnhafion and axis cylinder fragmentation Same as above, but more diffuse; epineurial thickening Demyelinization; axis cylinder fragmentation; intrafascicularedema and thickening af septa
0.1 0.25
Normal Epineurial thickening
0.001 0.01 0.1
Normal Normal Axis cylinder fragmentation; pale-staining myelin Same as above
1.0
* No changes in controls. i
"~
F i g u r e 1. Bodian stains of n e r v e s exposedtoHN2. A, Cross section showing pale staining and b r e a k - u p of many axis c y l i n d e r s (0.1 rag. per kg., pledget), x 128. B, Longitudinal section shovr axis cylinder f r a g m e n t a t i o n and dark a r e a s of h e m o r r h a g e (0.1 rag. p e r kg., injected), x 4 0 . C, C r o s s section showing gross i n t r a f a s c i c u l a r h e m o r r h a g e (1.0 rag. per kg., pledgeJ0, x 40.
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Drug
Dose (mg.lkg.) 0.001 O.005
HN2
0.010 0.100 1.000 0.001
A139
0.010 0.100 0.0001 0.001
Table 3. Histologic Changes in the Sciatic Nerve of the Dog Five Days Following Lower Limb Perfusion for 20 Minutes with Chemotherapeutic Agent*
Drug Demye[inization; eplneurial thickening Same as above, with axis cylinder frogmentation Same as above, plus intrafasclcular hemorrhage Same as above, but more marked Same as above, but more marked Demyelinizatlon; axis cylinder fragmentation; intrafascicular edema Same as above, but more marked Same as above, but more marked; intrafascicular hemorrhage Focal demyelinization Demyelinlzation; axis cylinder fragmentation Same as above, with intrafascicular edema Same as above, with more marked intrafascicular edema
0.010 0.100
Cytoxan
0.10 0.25
Normal Normal
0.001 0.010 0.100
Normal except for epineutial inflammation Same as above Demyelinizatlon; axis cylinder fragmentation Same as above
1.000
375
Histologic Nerve Changes
VLB
6-MPC
EFFECTS OF DRUGS ONNERVES
1962
Table 2. Histologlc Changes in the Sciatic Nerve of the Dog Five Days Following Subepineurial Injection of Chemotherapeutic Agent*
Dose (mg./kg.) 0.50 0.75 1.00
HN2 3.00 5.00 10.00
Histologic Nerve Changes Normal Normal Scattered demyelinlzation and displacement away from Schwann cell sheath Same as above Same as above, but less marked Same as above, plus poor axis cylinder staining
A139
10.00
Demyelinizatlon; focal axis cylinder fragmentation
VLB
1.00 4.00 6.00
Normal Early demyelinization Demyelinization; axis cylinder fragmentation
Cytoxan
40 80
Normal Normal
6-MPG
10.00
Early demyelinlzatlon
* No changes in controls. Drug dissolved throughout perfusate.
* No changes in controls.
epineurium. With higher concentrations, additional swelling, lateral migration and f r a g m e n t a t i o n of axis c y l i n d e r s w e r e noted, as well as intrafascicular hemorrhage and
edema. H N 2 perfusion under the stated experimental c o n d i t i o n s produced only scattered myelin changes except for additional early axis cylinder change with the highest dose {Figs. 1 and 2). A 1 3 9 administered by pledger or subepineurial injection produced myelin changes similar to those described above and, in addition, accumulation of a hyaline material in the intrafascicular areas and fragmentation of axis cylinders. Only the highest dose injected subepineurially caused intrafascicular hemorrhage. Perfusion with A 1 3 9 produced scattered myelin changes and spotty axis cylinder fragmentation (Fig. 3 A and B). V L B produced demyelinization inthe lower concentrations, while higher concentrations administered by pledger or subepineurial injection caused axis cylinder fragmentation and intrafascicular e d e m a with thickening of the sepia. Perfusion of this agent caused less prominent demyelinization and axis cylinder fragmentation {Fig. 3 C and D). Higher concentrations of 6 - M P G w e r e n e c essary to cause axis cylinder fragmentation
--N
F i g u r e 2. L F B - P A S stains of n e r v e s exposed to HN2. A, Longitudinal s e c t i o n showing v a c u o l i -
zation of myelin (0.01 rag. per kg., pledger) x 128. B, Cross section showing loss of rosette pattern of myelin and thin rim of myelin next to myelin sheath (0.I rag. per kg., injected),x 128.
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WOODHALL, MAHALEY, BOONE and H U N E Y C U T T k~ ~' i
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r,: ? F i g u r e 3. A, C r o s s s e c t i o n of Bodian s t a i n o f n e r v e i n j e c t e d with 0.1 mg. p e r kg. A139, showing h y a l i n e m a t e r i a l in i n t r a f a s c i c u l a r a r e a and s o m e l o s s of a x i s c y l i n d e r s , x 160. B, C r o s s s e c t i o n of L F B - P A S s t a i n of n e r v e i n j e c t e d with 0 . 0 0 i rag. p e r kg. A139, showing p a l e s t a i n i n g and d i s p l a c e m e n t away f r o m Schwann s h e a t h of m a n y m y e l i n s t r u c t u r e s , x 160. C, L o n g i t u d i n a l s e c t i o n of Bodian s t a i n of n e r v e inj e c t e d with 0.01 rag. p e r kg, VLB, showing v a c u o l i z a t i o n and f r a g m e n t a t i o n of a x i s c y l i n d e r s , x 160. D, C r o s s s e c t i o n of L F B - P A S s t a i n of n e r v e exposed b y p l e d g e t to 1.0 rag. p e r kg. VLB, showing i n t r a f a s c i c u l a r e d e m a and l o s s of m y e l i n , x 160. a n d d e m y e l i n i z a t i o n ( F i g . 4). C y t o x a n , in t h e concentrations tested, caused no histologic change in the sciatic nerve. Control injections and pledger applications with Ringer's solution and control perfusions without drugs c a u s e d no h i s t o l o g i c n e r v e c h a n g e s . O n l y nerve injection of drug was performed Table 4. HistologicChanges in the Sciatic Nerve of the Dog Five Days Following Subepineurial Iniection of PAM, Actinornycin D, and Methotrexate*
Drug
Dose (m~.Ikg.) 0.050 0.075
PAM 0.100 Actlnomycln D
0.003
0.006 Methotrexate
0.100 1.000
* No changes in controls.
Histologic Nerve Changes Vacuolization of myelin Myelin fragmentation; axis cylinder fragmentation; intrafasclcular edema and hemorrhage Same as above, but more marked Myelin fragmentation Myelin fragmentation; axis cylinder fragmentation Normal Vacuolizatlon of myelin, displaced away from sheath.
in the case of PAM, Actinomycin D and Methotrexate, and these results are summ a r i z e d i n T a b l e 4. W h e n 20 d a y s w e r e a l l o w e d to e l a p s e between nerve injection and histologic examination, the pathologic changes observed after injection of HN2, A139 and VLB were somewhat more severe, and there was a l y m p h o c y t i c i n f i l t r a t i o n ( F i g . 5). Nerve conduction studies revealed no alteration in the threshold stimulus necessary to transmit a nerve potential across the
injection site or in the conduction time across the injection site immediately or five days following subepineurial injection of Ringer's solution or Cytoxan. Changes in threshold and/or conduction time did occur five days (but not immediately ) after subepineurial injection of 0.I rag. per kg. of H N 2 , A139, V L B and 6 - M P G (Table 5). DISCUSSION These histologic and physiologic studies on the sciatic nerve of the dog were stimulated
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Table 5. Changes in Sciatic Nerve Conduction in Dogs Across Site of Subepineurial Injection of Chemotherapeutic Agent Five Days Previously*
Drug
Dose (rng./kg.)
HN2 A139 VLB
0.10 0.10 0.10
Cytoxon
0.25 0.10
6-MPG
Changes in Herve Conduction No conduction across lesion Threshold increased 4 volts
Threshold increased 2 volts; conduction time increased 1.38 x None Conduction time increased 1.16 x
* No conduction change in controls injected with sollne.
by s e v e r a l i n d i c a t i o n s of an e f f e c t of c e r t a i n chemotherapeutic agents, with some degree of s p e c i f i c i t y , upon c e n t r a l and p e r i p h e r a l n e r v e s . In a d d i t i o n to a r a t h e r r e m a r k a b l e relief of pain from tumor areas following regional perfusions, hearing loss has been noted following large intravenous doses of H N 2 , w i t h s p e c i a l t e c h n i q u e s e m p l o y e d to protect bone marrow. Peripheral neurop a t h i e s of the l o w e r e x t r e m i t i e s h a v e b e e n observed following regional pelvic perfusion w i t h HNZ a n d i n t r a v e n o u s t r e a t m e n t with VLB. Our experience w i t h 103 r e g i o n a l chemotherapeutic perfusions for neoplasms of the b r a i n , f a c e , p e l v i s , a b d o m e n and leg h a s b e e n t h a t n e r v e d y s f u n c t i o n i s r e l a t e d to
F i g u r e 4. C r o s s sections of n e r v e s injected with 0.1 mg. p e r kg. 6-1VIPG. x 160. A, Bodian stain, showing some swelling and loss of axis c y l i n d e r s . B, L F B - P A S stain, showing d i s p l a c e ment of m y e l i n away f r o m the Schwann sheath and s c a t t e r e d loss of r o s e t t e p a t t e r n .
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F i g u r e S. Bodian stains 20 days a f t e r injection of 0.1 rag. p e r kg. of drugs, x 160. A, HN2, c r o s s section, showing s e v e r e loss of axis c y l i n d e r s andlymphocytic infiltration. B, A139, longitudinal section, showing h e m o r r h a g e , axis c y l i n d e r f r a g m e n t a t i o n and lymphocytes. C, VLB, c r o s s section, showing i n t r a f a s c i c u l a r edema, loss of axis c y l i n d e r s and Iymphocytes.
378
WOODHALL, M A H A L E Y , BOONE a n d H U N E Y C U T T Table 6. Counts Per Minute (CPM) of C 14 in CNS 45 Minutes After Intravenous Iniection of 15#c. of HN2-C 14 (0.75 mg./kg.)
Sample Frontal" Parietal Occipital Cerebellum Hypophysis Midbrain Cervical cord Cranial nerve I II III V VI VII Vlll IX, X, Xl
CPM/gm. 180 164 168 160 797 179 103 341 462 0 268 0 1027 3333 881
high doses of chemotherapeutic agents, either concentrated throughout smaller volumes of extracorporeal perfusate or injected directly into the arterial inflow catheter. O u r first investigations were directed toward postperfusion cranial nerve dysfunction, involving primarily nerves VIII, VII, V and VI. Cranial nerve localization of H N 2 injected intravenously in dogs has been investigated using chloroethyl-C 14 labeled H N 2 . Iz There was a selective localization in the cranial nerves, especially the eighth, as well as in the hypophysis (Table 6). Subsequent studies with cl4-1abeled Cytoxan have shown highest radioactivity in the hypophysis but none inthe cranial nerves (Table 7). These data correlate with the higher incidence of cranial nerve dysfunction following H N 2 treatment in patients. The studies reported here concern primarily histologic changes inthe sciatic nerve of the dog following exposure to anticancer agents by topical application, subepineurial
TabTe 7. Counts Per Minute (CPM) of C 14 in CNS Two Hours After Intravenous Injection of 15 Fe. of Cytoxan - C 14 (40 mg./kg.)
Sample Frontal Occipital Cerebellum Hypophysis Midbrain Cervical cord Cranial nerves I - XII
CPM/gm. 10 10 19 197 17 12 0
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i n j e c t i o n o r r e g i o n a l l i m b p e r f u s i o n . No changes occurred in controls or in Cytoxanexposed nerves. The lowest concentrations of H N 2 , A139, V L B , PAM, Actinomycin D, Methotrexate and 6 - M F G caused alteration in the staining characteristics o f the myelin, which b e c a m e swollen, vacuolated, thinned and displaced f r o m the S c h w a n n sheath. Higher concentrations produced additional c h a n g e s , w i t h v a r i a t i o n s w i t h e a c h d r u g . HtN2 caused marked axis cylinder swelling and eventual fragmentation as well as intrafascicular hemorrhage and edema. A139 and V L B also caused axis cylinder fragmentation, but they also produced an unusual intrafascic ular deposition of hyaline material rarely associated with hemorrhage. The myelin and axis cylinder changes with 6-~/LIDG w e r e less remarkable. Vasa n e r v o r u m histologically appeared normal. Corresponding nerve conduction changes w e r e noted with HN2, A139, VLB and 6-MI~ but not with Cytoxan or controls. The anticancer agents that produced the most remarkable changes w h e n applied locally caused relatively benign histologic changes following regional leg perfusion. E a c h drug was dissolved in the entire perfusate prior to onset of perfusion. It has subsequently been found that H N 2 loses its activity rapidly in vitro in blood o r p l a s m a 14 and that H N 2 and A 1 3 9 are rapidly cleared f r o m the blood in vivo. 13.14 The lack of remarkable nerve changes following perfusion with these dosages was felt to be due to in vitro deterioration of drug in the perfusate and to rapid uptake of drug by all tissues of the perfused leg. T o investigate the first of these two possibilities, deterioration of drug in vitro in the perfusate was reduced by injecting H N 2 directly into the arterial inflow catheter after perfusion was established (Table 8). This Table 8. Histolagic Changes in the Sciatic Nerve of the Dog Five Days Following HN2 Injection in the Arterial Inflow Catheter During Regional Lower Limb Perfuslon for 20 Minutes
Dose (mg./kg.) 1.0 2.5 5.0
HistologicNerve Changes Demye[ini zation Demyelinization; axis cylinder fragmentation; epineurial thickening Demyelinization; axis cylinder fragmentation; epineurial thickening
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F i g u r e 6. C r o s s sections of n e r v e s taken f r o m legs p e r f u s e d withHN2 d i s s o l v e d throughout the p e r f u s a t e or injected in the a r t e r i a l inflow c a t h e t e r , x 160. A, L F B - P A S stain, 5.0 rag. p e r kg. d i s s o l v e d in p e r f u s a t e , showing v e r y l i t t l e a l t e r a t i o n of r o s e t t e p a t t e r n of myelin. B, Bodian stain, 3.0 mg. per kg. d i s s o l v e d in p e r f u s a t e , showing r e l a t i v e l y n o r m a l axis c y l i n d e r s . C, L F B - P A S ' s t a i n , 2.5 rag. per kg. injected in a r t e r i a l inflow c a t h e t e r , resulting in s e v e r e loss o s D, Bodian stain, 2.5 rag. per kg. injected in a r t e r i a l inflow c a t h e t e r , showing i n t r a f a s c i c u l a r edema and loss os axis c y l i n d e r s . t e c h n i q u e of H N 2 a d m i n i s t r a t i o n r e s u l t e d in m o r e s t r i k i n g p a t h o l o g i c c h a n g e s than had b e e n s e e n w h e n HN2 w a s d i s s o l v e d t h r o u g h o u t the p e r f u s a t e ( F i g . 6). T o i n v e s t i g a t e t h e s e c o n d p o s s i b i l i t y , c h l o r o e t h y 1 - C 14 l a b e l e d HN2 w a s p e r f u s e d to t h e l e g s o f two d o g s . T h i r t y m i n u t e s following p e r f u s i o n , the s c i a t i c n e r v e , f e m o r a l a r t e r y , f e m o r a l v e i n and a p o r t i o n of m u s c l e f r o m t h e p e r f u s e d a n d f r o m the o p p o s i t e l e g s w e r e e x a m i n e d for the p r e s e n c e o f r a d i o a c t i v i t y ( T a b l e 9). T h e t i s s u e s from the perfused leg had the highest concent r a t i o n s a s c o m p a r e d to t h o s e o f t h e t o t a l b o d y , Table 9. Counts Per Minute (CPM) of C 14 in Tissue
Samples 30 Minutes After Regional Lower Limb Perfusion with 15/~c. of HN2- C 14
Tissue Samples Femoral artery Femoral vein Muscle Sciatic nerve
CPM/gm. Perfused leg Opposite/eg 1554 647
51 0
62 13
9 0
b u t t h e c o n c e n t r a t i o n in t h e n e r v e w a s l o w e s t . T h u s , it w o u l d s e e m t h a t b o t h in v i t r o d r u g d e t e r i o r a t i o n in the p e r f u s a t e and uptake of d r u g by s e v e r a l t i s s u e s o t h e r t h a n n e r v e o c c u r r e d in t h e e x p e r i m e n t a l l i m b p e r f u s i o n s ( T a b l e 3). Q u a n t i t a t i v e l y , n e r v e t i s s u e d i d not selectively, concentrate HN2, when compared with muscle, following experimental regional limb perfusion. Therefore the pcripheral neuropathies seen following perf u s i o n s in p a t i e n t s probably represent a q u a l i t a t i v e t i s s u e s u s c e p t i b i l i t y in t h e p r e s ence of a relatively high local concent r a t i o n of the a n t i t u m o r substance, while neuropathies following intravenous drug administration may also reflect this peculiar sensitivity. Thes e studies indicate a primary alteration in m y e l i n s t r u c t u r e , a s s o c i a t e d w i t h a n e r v e c o n d u c t i o n d e f e c t . The s u s c e p t i b i l i t y of m y e l i n m a y be d e p e n d e n t upon the lipid s o l u b i l i t y of HN2, which probably enhances its entrance into cells, l's'? The hemorrhage and edema of the i n t r a f a s c i c u l a r spaces characteristic of h i g h e r d o s a g e s a r e i d e n t i c a l with the
380
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WOODIlALL, MAIIALEY, BOONE
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F i g u r e 7. Sciatic n e r v e of patient six days following pelvic perfusion with 60 rag. HN2. A, Longitudinal section, Bodian stain, showing loss of axis c y l i n d e r s , x 160. B, C r o s s section, L F B - P A S stain, showing pale staining of m]~elinand d i s p l a c e m e n t a w a y f r o m Schwann sheath, x 160. C, C r o s s section, L F B - P A S stain, showing subepineurial h e m o r r h a g e , x 64. vascular necrosis described in earlier s t u d i e s 4 and in the b r a i n following c a r o t i d a r t e r y i n j e c t i o n . S, 9 T h e s a m e c o n c e p t s w o u l d a p p e a r valid for the r e m a i n i n g a c t i v e a n t i tumor systems that were studied. The postperfusion peripheral neuropathy of a p a t i e n t h a s b e e n s t u d i e d a t a u t o p s y . P e l v i c p e r f u s i o n w a s d o n e w i t h 60 rag. of HNZ i n j e c t e d d i r e c t l y i n t o t h e a r t e r i a l i n f l o w catheter. Bilateral sciatic nerve paralysis d e v e l o p e d on the day following p e r f u s i o n , five days before death. Demyelinization, axis cylinder fragmentation, edema and focal hemo r r h a g e was found in the s c i a t i c n e r v e s {Fig. 7). These observations do n o t e x p l a i n the m a r k e d r e l i e f of p a i n t h a t f o l l o w s p e r f u s i o n of m a l i g n a n t t u m o r s w i t h c o m m o n l y u s e d a l k y l a t i n g a g e n t s i n the l a c k of a n y o b v i o u s neurologic changes, More sophisticated nerve fiber conduction studies might elucidate this p h e n o m e n o n . One p a t i e n t with tic d o u l o u r e u x and bundle-branch heart block has been perf u s e d t h r o u g h the e x t e r n a l c a r o t i d a r t e r y - i n t e r n a l j u g u l a r v e i n w i t h A 1 3 9 . R e l i e f of tic p a i n has b e e n o b t a i n e d for nine m o n t h s with
concomitant but mild radiomimetic skin c h a n g e s a n d s l i g h t l o s s of p a i n d i s c r i m i n a tion. Adequate dosage titration may permit an e x t e n s i o n of this p r o v o c a t i v e t e c h n i q u e of pain control. SUMMARY S t u d i e s a r e d e s c r i b e d w h e r e i n the s c i a t i c n e r v e of the dog w a s e x p o s e d to c h e m o t h e r a p e u t i c a g e n t s by t o p i c a l a p p l i c a t i o n , s u b e p i n e u r i a l i n j e c t i o n o r r e g i o n a l l i m b pe.rfusion. Histologic and nerve potential conduction changes are reported. The relationof t h e s e f i n d i n g s to v a r i o u s neuropathies observed clinically following treatment with t h e s e a g e n t s is d i s c u s s e d . REFERENCES I. Boyland, E.: The toxicity of a l k y l - b i s ({~chloroethyl) a m i n e s and of the products of their reaction with water. Brit. J. Pharmacol., l: Z47, 1946. 2. Conrad, M. E., J r . , and Crosby, W. H.: k{assive nitrogen m u s t a r d therapy in Hodgkin's disease with protection of bone m a r r o w by tourniquets. Blood, 16: 1089, 1960.
]SR -
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3. C r e e c h , O. J . , Ryan, i%. F . , and K r e m e n t z , E. T.: R e g i o n a l c h e m o t h e r a p y by i s o l a t e d p e r f u s i o n in t h e t r e a t m e n t o f m e l a n o m a of the e x t r e m i t i e s . P l a s t . & R e c o n s t r u c t . S u r g . , 28: 333, 1961. 4. D a n i e l l i , J . F . : C e l l P h y s i o l o g y and P h a r m a c o l o g y , New York, E l s e v i e r P u b l i s h i n g Co., Inc., 1950. 5. F e l l , H. B., and A l l s o p p , C. B.: T i s s u e c u l t u r e e x p e r i m e n t s on the b i o l o g i c a l a c t i o n o f m e t h y l b i s ( ~ - c h l o r o e t h y l ) a m i n e and its h y d r o l y s i s p r o d u c t s . C a n c e r Ftes., 9: 238, 1949. 6. F r e n c h , J . D., W e s t , P. M., v o n A m e r o n g e n , F . K., and M a g o u n , H. W.: E f f e c t s of i n t r a c a r o t i d a d m i n i s t r a t i o n of n i t r o g e n m u s t a r d on n o r m a l b r a i n and b r a i n t u m o r s . J . N e u r o s u r g . , 9: 378, 1952. 7. G i l m a n , A., a n d P h i l l i p s , F . S.: T h e b i o l o g i c a l a c t i o n s and t h e r a p e u t i c a p p l i c a t i o n o f f i - c h l o r o e t h y l a m i n e s and s u l f i d e s . S c i e n c e , 103:409, 1946. 8. H e r t z , R.: T h e r a p e u t i c t r i a l s with v i n c a l e u k o b l a s t i n e in p a t i e n t s with M e t h o t r e x a t e - r c s i s t a n t cho r i o c a rc i n o m a . C a n a d i a n Cane e r C o n f e r e n c e, N e w York, A c a d e m i c P r e s s , 1961, p. 383. 9. K1opp, C. T., A1ford, T. S., B a t e m a n , J., B e r r y , G. N., and W i n s h i p , T.: F r a c t i o n a t e d intra-arterial cancer c h e m o t h e r a p y with m e t h y l b i s (fl- chlo r o e t h y l ) a m i n e h y d r o c h l o r i d e . Ann. S u r g . , 132: 811, 1950. 10. Lawrence, W., Jr., Kuehn, P., Masle, E. T., and Miller, D. G.: A n abdominal tourniquet for regional chemotherapy. J. Surg. Res., I: 142, 1961. 11. L y o n s , W. R., and Woodhall, B.: A t l a s of P e r i p h e r a l N e r v e I n j u r i e s . P h i l a d e l p h i a , W. B. S a u n d e r s C o . , 1949. lZ. M a h a l e y , I%{. S., J r . , H u n e y c u t t , H., Boone, S., and Woodhall, B.: L o c a l i z a t i o n of m e t h y l bis (2-chloroethyl-1, 2-C14)amine hydrochloride in n e r v o u s t i s s u e a f t e r i n t r a v e n o u s i n j e c t i o n o r r e g i o n a l c e r e b r a l p e r f u s i o n in d o g s . C a n c e r C h e m o t h e r . R e p . , 11:29, 1961. 13. M a h a l e y , M. S., J r . , M a h a l e y , J a n e , and Woodhall, B . : B i o a s s a y o f p l a s m a l e v e l s o f A 1 3 9 and m a n n i t o l m u s t a r d f o r u s e in d e t e r m i n i n g blood l e v e l s d u r i n g r e g i o n a l b r a i n c a n c e r p e r f u s i o n s . C a n c e r C h e m o t h e r . R e p . , 11: 33, 1961.
EFFECTS
OF D R U G S ON N E R V E S
381
14.
M a h a l e y , M. S., J r . , and WoodhaI1, 13.: P l a s m a l e v e l s of a l k y l a t i n g a g e n t s d u r i n g r e g i o n a l c h e m o t h e r a p e u t i c p e r f u s i o n s . J. Surg. Res., 1: 285, 1961.
15.
M a i m , J. Ft., H e r t e r , F . P., F r i c k , H. C., Long, M. E., and D e m e t z , A.: E v a l u a t i o n o f i s o l a t e d p e r f u s i o n c h e m o t h e r a p y in the t r e a t m e n t of a d v a n c e d m a l i g n a n t d i s e a s e . S u r g . F o r u m , I0:743, 1959.
16. Shingleton, W. W., Parker, R.T.,andMahaley, S.: Abdominal perfusion for cancer chemotherapy. Ann. Surg., 152: 583, 1960. 17. Sholes, D. ~I.: Pelvic perfusion with nitrogen mustard for cancer: a neurological complication. A m . J. Obst. & Gynec., 80:481, 1960. 18. Strelinger, A. L., Gilbert, L., and Shoshkes, M.: Extracorporeal regional perfusion with nitrogen mustard for the palliation of amalignant tumor. J. N e w a r k Beth Israel Hosp., II: 79, 1960. 19. Vaitkevicius, V.K., Talley, R. W., and Brennan, M. J.: Cytological and clinical observations during vincaleukoblastine therapy of disseminated cancer. Proc. A m . A. Cancer Ftes., 3: 275, 1961. 20. %Vill, J. J., Korst, D. 1%., Monto, R. W., and Hurley, J. D.: Phase II evaluation of vincaleukoblastine. A study by the MidwestCooperative Chemotherapy Group. Proc. A m . A. Cancer Res., 3:Z78, 1961. 21. Woodhall, B., Hall, K., Mahaley, S., Jr., and Jackson, J.: Chemotherapy of brain cancer: experimental and clinical studies in localized hypothermic cerebral perfusion. Ann. Surg., 150: 640, 1959. 22. Woodhall, B., Pickrell, K. L., Georgiade, N. G., Mahaley, M. S., Jr., and Dukes, H. T.: Effect of hyperthermia upon cancer chemotherapy; alSplication to external cancers of head and face structures. Ann. Surg., 151: 750, 1960. 23. Young, W. G., Jr., Lesage, A. M., Dillon, M. L., Lee, J. M., Callaway, H. A., Jr., and Reeves, J. %V.: Chemotherapy of intrathoracic neoplasms employing differential pelvic perfusion hypotherrnia. Ann. Surg., 154: 372, 1961.
The first indication that specific changes might occur in peripheral nerve tissue following exposure to c e r t a i n a n t i t u m o r a g e n t s d e v e l o p e d f r o m the o b s e r v a t i o n t h a t r e l i e f of p a i n f r o m t h e t u m o r a r e a o c curred after regional chemotherapeutic perf u s i o n , ls'm'2''22 P a t i e n t s t r e a t e d w i t h l a r g e intravenous dosages ofnitrogenmustard, with s p e c i a l t e c h n i q u e s f o r the p r o t e c t i o n of b o n e marrow, also developed focal cranial nerve d e f i c i t s i n the f o r m of h e a r i n g l o s s . z. t0,23 S u b s e q u e n t l y , n i t r o g e n m u s t a r d - - C 14 i n j e c t e d i n t r a v e n o u s l y i n d o g s w a s f o u n d to l o c a l i z e s e l e c t i v e l y in the eighth n e r v e , a m o n g other c r a n i a l n e u r a l s t r u c t u r e s . Iz P e r i p h e r a l n e u r o p a t h i e s of the l o w e r e x t r e m i t i e s h a v e b e e n r e p o r t e d a s a s e q u e l of r e g i o n a l p e l v i c p e r fusion with mustards3"17 and intravenous therapy with v i n c a l e u k o b l a s t i n e , s. 19.20 T h e s e d o c u m e n t e d e v i d e n c e s of n e u r o t o x i c drug a c t i o n in the p e r i p h e r a l and c r a n i a l nerve systems, together with earlier evidence c o n c e r n e d with the c e n t r a l n e r v o u s s y s t e m , s s t i m u l a t e d t h i s s t u d y of the e f f e c t of c h e m o t h e r a p e u t i c a g e n t s u p o n t h e s c i a t i c n e r v e of the dog. MATERIALS AND METHODS The drugs used in these experiments were: methyl-bis (fl-chloroethyl)-amine hydrochlor i d e (HN2); 2 , 5 - d i e t h y l e n i m x n o - 3 , 6 - b i s ( 2 - m e t h o x y e t h o x y ) - 1 , 4 - b e n z o q u i n o n e (A139); v i n c a l e u k o b l a s t i n e s u l f a t e ( V L B ) ; 2 H - l , 3, 2oxazaphosphorine, 2-bis(chloroethy1) amino-
F r o m the Division of N e u r o s u r g e r y , the Duke U n i v e r s i t y Medical School, Durham, North Ca rolina. Supported by U.S.P.H.S. Grants # M 2109, H 3582 and CY 4751, from the National Institutes of Health, Bethesda, Maryland. Submitted for publication December 26, 1961. ]$R -
V o l . Ii, No. 6 - N o v e m b e r , 1962
BOONE,
M . D.,
tetrahydro-2-oxide (Cytoxan); and 6-purine f l - D - g l u c o t h i o p y r a n o s i d e (6-1viPG). L e s s i n t e n s i v e s t u d i e s w e r e c a r r i e d out with p - b i s (2-chloroethyl) a m i n o p h e n y l a l a n i n e (PAM), Actinomycin D and Methotrexate. Each drug was dissolved in Ringer's solution (except PAM, which was dissolved in propylene glycol). Each sciatic nerve was exposed in the t h i g h b y s e p a r a t i n g t h e b i c e p s f e m o r i s and the s e m i t e n d i n o s u s m u s c l e s . I n t h e f i r s t g r o u p of e x p e r i m e n t s a f e l t p l e d g e t 1 c m . s q u a r e w a s p l a c e d u p o n the epineurium. The pledget was saturated with 0.1 c c . of the d r u g s o l u t i o n ( e x p e r i m e n t a l ) or R i n g e r ' s s o l u t i o n (control). A f t e r five m i n u t e s t h e p l e d g e t w a s r e m o v e d a n d the i n c i s i o n w a s c l o s e d . I n t h e s e c o n d g r o u p the d r u g o r d i l u e n t (0.1 c c . ) w a s i n j e c t e d b e n e a t h the e p i n e u r i u m . I n t h e t h i r d g r o u p a l o w e r l i m b w a s r e g i o n a l l y p e r f u s e d f o r 20 m i n u t e s v i a the f e m o r a l a r t e r y a n d v e i n . T h e d r u g w a s m i x e d u n i f o r m l y i n t h e p e r f u s a t e , 500 c c . of w h o l e b l o o d a t 37 ~ C. T h e e x t r a c o r poreal circuit consisted of a m o d i f i e d Travenol bubble oxygenator, a Harrison heat exchanger, a bubble trap, a rotameter flowm e t e r and a S i g m a m o t o r pump. P a r a m e t e r s m e a s u r e d w e r e systemic arterial pressure, perfusion arterial pressure, perfusate temperature and perfusion flow. Following perfusion the vessels w e r e repaired withS-0 arterial silk. A control perfusion was done in one dog with no chemotherapeutic agent in the perfusate. Five days after drug administration, each sciatic nerve w a s taken for h i s t o l o g i c e x a m i n a t i o n ; i n s o m e c a s e s 20 d a y s w e r e a l l o w e d to e l a p s e b e t w e e n n e r v e injection and histologic examination. Cross and longitudinal sections were stained with hematoxylin and eosin, Bodian's solution, luxol-fast-blue and periodic acid--Schiff stains. Nerve conduction studies w e r e p e r f o r m e d across the site of subepineurial injection of 373
374
WOODHALL, MAHALEY, BOONE a n d [ [ U N E Y C U T T
JSR
Drug
Histologic Nerve Changes
0.001 0.005
Demyelinization~ thickening of epineurium Demyelinization~,. thickening of eplneurium, migration of axis cylinders laterally DemyellnizationI thickening of eplneurium, localized intrafascicular hemorrhage and edema; endoneurlal thickening; axis ay[{nder swe[llng and disappearance Same as above, with more diffuse changes and mare marked endoneurial thicken{ng Same as above
HN2 0.1 1.0 A139
0.001 0.01 0.1 0.0001 0.001 0.01
VLB 0.1
The histologic changes noted following drug administration bypledget, subepineurial injection and perfusion are listed in Tables i , Z a n d 3, r e s p e c t i v e l y (the h i s t o l o g i c c h a r a c t e r i s t i c s of n o r m a l p e r i p h e r a l n e r v e s h a v e been described It). The smallest concentrat i o n of HN2 a d m i n i s t e r e d by pledger or subepineurial injection caused swelling, pale s t a i n i n g a n d d i s o r g a n i z a t i o n of t h e m y e l i n , a s w e l l a s a f i b r o b l a s t i c r e s p o n s e a l o n g the
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1962
Dose (mg./kg.)
0.01
RESULTS
.
November,
Table 1, Histologlo Changes in the Sciatic Nerve of the Dog Five Days Following Five-Minute Pledget Application of Chemotherapeutic Agents*
drug or Ringer's solution. Abipolarplatinum stimulating electrode was placed onthe nerve 6 c m . p r o x i m a l to t h e i n j e c t i o n s i t e , a n d b i p o l a r p l a t i n u m r e c o r d i n g e l e c t r o d e s (5 m m . a p a r t ) w e r e p l a c e d 2 c m . d i s t a l to the i n j e c t i o n 9 s i t e . T h e n e r v e w a s s t i m u l a t e d (3 v o l t s ) w i t h a G r a s s s t i m u l a t o r ( s i n g l e , m o n o p h a s i c 0.01 msec. impulses) and isolation unit, andnerve potentials were recorded distally by a Grass P - 6 p r o b e a n d D . C . a m p l i f i e r , fed i n t o a DuMont oscilloscope and Grass Kymograph camera. Records were taken before nerve injection, immediately after injection and five days later. Drug dosages that produced histologic nerve changes were chosen for conduction studies.
, 7
-- V o l . II, N o . 6 -
1.0
Cytoxan
6-MPG
Demyelinization; intrafascicular edema Same as above, but more marked Same as above, plus axis cylinder flagmentation Normal Normal Localized demyelJnhafion and axis cylinder fragmentation Same as above, but more diffuse; epineurial thickening Demyelinization; axis cylinder fragmentation; intrafascicularedema and thickening af septa
0.1 0.25
Normal Epineurial thickening
0.001 0.01 0.1
Normal Normal Axis cylinder fragmentation; pale-staining myelin Same as above
1.0
* No changes in controls. i
"~
F i g u r e 1. Bodian stains of n e r v e s exposedtoHN2. A, Cross section showing pale staining and b r e a k - u p of many axis c y l i n d e r s (0.1 rag. per kg., pledget), x 128. B, Longitudinal section shovr axis cylinder f r a g m e n t a t i o n and dark a r e a s of h e m o r r h a g e (0.1 rag. p e r kg., injected), x 4 0 . C, C r o s s section showing gross i n t r a f a s c i c u l a r h e m o r r h a g e (1.0 rag. per kg., pledgeJ0, x 40.
JSR -
V o l . 11, N o . 6 -
November,
Drug
Dose (mg.lkg.) 0.001 O.005
HN2
0.010 0.100 1.000 0.001
A139
0.010 0.100 0.0001 0.001
Table 3. Histologic Changes in the Sciatic Nerve of the Dog Five Days Following Lower Limb Perfusion for 20 Minutes with Chemotherapeutic Agent*
Drug Demye[inization; eplneurial thickening Same as above, with axis cylinder frogmentation Same as above, plus intrafasclcular hemorrhage Same as above, but more marked Same as above, but more marked Demyelinizatlon; axis cylinder fragmentation; intrafascicular edema Same as above, but more marked Same as above, but more marked; intrafascicular hemorrhage Focal demyelinization Demyelinlzation; axis cylinder fragmentation Same as above, with intrafascicular edema Same as above, with more marked intrafascicular edema
0.010 0.100
Cytoxan
0.10 0.25
Normal Normal
0.001 0.010 0.100
Normal except for epineutial inflammation Same as above Demyelinizatlon; axis cylinder fragmentation Same as above
1.000
375
Histologic Nerve Changes
VLB
6-MPC
EFFECTS OF DRUGS ONNERVES
1962
Table 2. Histologlc Changes in the Sciatic Nerve of the Dog Five Days Following Subepineurial Injection of Chemotherapeutic Agent*
Dose (mg./kg.) 0.50 0.75 1.00
HN2 3.00 5.00 10.00
Histologic Nerve Changes Normal Normal Scattered demyelinlzation and displacement away from Schwann cell sheath Same as above Same as above, but less marked Same as above, plus poor axis cylinder staining
A139
10.00
Demyelinizatlon; focal axis cylinder fragmentation
VLB
1.00 4.00 6.00
Normal Early demyelinization Demyelinization; axis cylinder fragmentation
Cytoxan
40 80
Normal Normal
6-MPG
10.00
Early demyelinlzatlon
* No changes in controls. Drug dissolved throughout perfusate.
* No changes in controls.
epineurium. With higher concentrations, additional swelling, lateral migration and f r a g m e n t a t i o n of axis c y l i n d e r s w e r e noted, as well as intrafascicular hemorrhage and
edema. H N 2 perfusion under the stated experimental c o n d i t i o n s produced only scattered myelin changes except for additional early axis cylinder change with the highest dose {Figs. 1 and 2). A 1 3 9 administered by pledger or subepineurial injection produced myelin changes similar to those described above and, in addition, accumulation of a hyaline material in the intrafascicular areas and fragmentation of axis cylinders. Only the highest dose injected subepineurially caused intrafascicular hemorrhage. Perfusion with A 1 3 9 produced scattered myelin changes and spotty axis cylinder fragmentation (Fig. 3 A and B). V L B produced demyelinization inthe lower concentrations, while higher concentrations administered by pledger or subepineurial injection caused axis cylinder fragmentation and intrafascicular e d e m a with thickening of the sepia. Perfusion of this agent caused less prominent demyelinization and axis cylinder fragmentation {Fig. 3 C and D). Higher concentrations of 6 - M P G w e r e n e c essary to cause axis cylinder fragmentation
--N
F i g u r e 2. L F B - P A S stains of n e r v e s exposed to HN2. A, Longitudinal s e c t i o n showing v a c u o l i -
zation of myelin (0.01 rag. per kg., pledger) x 128. B, Cross section showing loss of rosette pattern of myelin and thin rim of myelin next to myelin sheath (0.I rag. per kg., injected),x 128.
376
WOODHALL, MAHALEY, BOONE and H U N E Y C U T T k~ ~' i
,
*
'~ o'~.:~" .......
'
~
JSR -
V o l . !!, N o . 6 -
November,
1962
'
r,: ? F i g u r e 3. A, C r o s s s e c t i o n of Bodian s t a i n o f n e r v e i n j e c t e d with 0.1 mg. p e r kg. A139, showing h y a l i n e m a t e r i a l in i n t r a f a s c i c u l a r a r e a and s o m e l o s s of a x i s c y l i n d e r s , x 160. B, C r o s s s e c t i o n of L F B - P A S s t a i n of n e r v e i n j e c t e d with 0 . 0 0 i rag. p e r kg. A139, showing p a l e s t a i n i n g and d i s p l a c e m e n t away f r o m Schwann s h e a t h of m a n y m y e l i n s t r u c t u r e s , x 160. C, L o n g i t u d i n a l s e c t i o n of Bodian s t a i n of n e r v e inj e c t e d with 0.01 rag. p e r kg, VLB, showing v a c u o l i z a t i o n and f r a g m e n t a t i o n of a x i s c y l i n d e r s , x 160. D, C r o s s s e c t i o n of L F B - P A S s t a i n of n e r v e exposed b y p l e d g e t to 1.0 rag. p e r kg. VLB, showing i n t r a f a s c i c u l a r e d e m a and l o s s of m y e l i n , x 160. a n d d e m y e l i n i z a t i o n ( F i g . 4). C y t o x a n , in t h e concentrations tested, caused no histologic change in the sciatic nerve. Control injections and pledger applications with Ringer's solution and control perfusions without drugs c a u s e d no h i s t o l o g i c n e r v e c h a n g e s . O n l y nerve injection of drug was performed Table 4. HistologicChanges in the Sciatic Nerve of the Dog Five Days Following Subepineurial Iniection of PAM, Actinornycin D, and Methotrexate*
Drug
Dose (m~.Ikg.) 0.050 0.075
PAM 0.100 Actlnomycln D
0.003
0.006 Methotrexate
0.100 1.000
* No changes in controls.
Histologic Nerve Changes Vacuolization of myelin Myelin fragmentation; axis cylinder fragmentation; intrafasclcular edema and hemorrhage Same as above, but more marked Myelin fragmentation Myelin fragmentation; axis cylinder fragmentation Normal Vacuolizatlon of myelin, displaced away from sheath.
in the case of PAM, Actinomycin D and Methotrexate, and these results are summ a r i z e d i n T a b l e 4. W h e n 20 d a y s w e r e a l l o w e d to e l a p s e between nerve injection and histologic examination, the pathologic changes observed after injection of HN2, A139 and VLB were somewhat more severe, and there was a l y m p h o c y t i c i n f i l t r a t i o n ( F i g . 5). Nerve conduction studies revealed no alteration in the threshold stimulus necessary to transmit a nerve potential across the
injection site or in the conduction time across the injection site immediately or five days following subepineurial injection of Ringer's solution or Cytoxan. Changes in threshold and/or conduction time did occur five days (but not immediately ) after subepineurial injection of 0.I rag. per kg. of H N 2 , A139, V L B and 6 - M P G (Table 5). DISCUSSION These histologic and physiologic studies on the sciatic nerve of the dog were stimulated
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Table 5. Changes in Sciatic Nerve Conduction in Dogs Across Site of Subepineurial Injection of Chemotherapeutic Agent Five Days Previously*
Drug
Dose (rng./kg.)
HN2 A139 VLB
0.10 0.10 0.10
Cytoxon
0.25 0.10
6-MPG
Changes in Herve Conduction No conduction across lesion Threshold increased 4 volts
Threshold increased 2 volts; conduction time increased 1.38 x None Conduction time increased 1.16 x
* No conduction change in controls injected with sollne.
by s e v e r a l i n d i c a t i o n s of an e f f e c t of c e r t a i n chemotherapeutic agents, with some degree of s p e c i f i c i t y , upon c e n t r a l and p e r i p h e r a l n e r v e s . In a d d i t i o n to a r a t h e r r e m a r k a b l e relief of pain from tumor areas following regional perfusions, hearing loss has been noted following large intravenous doses of H N 2 , w i t h s p e c i a l t e c h n i q u e s e m p l o y e d to protect bone marrow. Peripheral neurop a t h i e s of the l o w e r e x t r e m i t i e s h a v e b e e n observed following regional pelvic perfusion w i t h HNZ a n d i n t r a v e n o u s t r e a t m e n t with VLB. Our experience w i t h 103 r e g i o n a l chemotherapeutic perfusions for neoplasms of the b r a i n , f a c e , p e l v i s , a b d o m e n and leg h a s b e e n t h a t n e r v e d y s f u n c t i o n i s r e l a t e d to
F i g u r e 4. C r o s s sections of n e r v e s injected with 0.1 mg. p e r kg. 6-1VIPG. x 160. A, Bodian stain, showing some swelling and loss of axis c y l i n d e r s . B, L F B - P A S stain, showing d i s p l a c e ment of m y e l i n away f r o m the Schwann sheath and s c a t t e r e d loss of r o s e t t e p a t t e r n .
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F i g u r e S. Bodian stains 20 days a f t e r injection of 0.1 rag. p e r kg. of drugs, x 160. A, HN2, c r o s s section, showing s e v e r e loss of axis c y l i n d e r s andlymphocytic infiltration. B, A139, longitudinal section, showing h e m o r r h a g e , axis c y l i n d e r f r a g m e n t a t i o n and lymphocytes. C, VLB, c r o s s section, showing i n t r a f a s c i c u l a r edema, loss of axis c y l i n d e r s and Iymphocytes.
378
WOODHALL, M A H A L E Y , BOONE a n d H U N E Y C U T T Table 6. Counts Per Minute (CPM) of C 14 in CNS 45 Minutes After Intravenous Iniection of 15#c. of HN2-C 14 (0.75 mg./kg.)
Sample Frontal" Parietal Occipital Cerebellum Hypophysis Midbrain Cervical cord Cranial nerve I II III V VI VII Vlll IX, X, Xl
CPM/gm. 180 164 168 160 797 179 103 341 462 0 268 0 1027 3333 881
high doses of chemotherapeutic agents, either concentrated throughout smaller volumes of extracorporeal perfusate or injected directly into the arterial inflow catheter. O u r first investigations were directed toward postperfusion cranial nerve dysfunction, involving primarily nerves VIII, VII, V and VI. Cranial nerve localization of H N 2 injected intravenously in dogs has been investigated using chloroethyl-C 14 labeled H N 2 . Iz There was a selective localization in the cranial nerves, especially the eighth, as well as in the hypophysis (Table 6). Subsequent studies with cl4-1abeled Cytoxan have shown highest radioactivity in the hypophysis but none inthe cranial nerves (Table 7). These data correlate with the higher incidence of cranial nerve dysfunction following H N 2 treatment in patients. The studies reported here concern primarily histologic changes inthe sciatic nerve of the dog following exposure to anticancer agents by topical application, subepineurial
TabTe 7. Counts Per Minute (CPM) of C 14 in CNS Two Hours After Intravenous Injection of 15 Fe. of Cytoxan - C 14 (40 mg./kg.)
Sample Frontal Occipital Cerebellum Hypophysis Midbrain Cervical cord Cranial nerves I - XII
CPM/gm. 10 10 19 197 17 12 0
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i n j e c t i o n o r r e g i o n a l l i m b p e r f u s i o n . No changes occurred in controls or in Cytoxanexposed nerves. The lowest concentrations of H N 2 , A139, V L B , PAM, Actinomycin D, Methotrexate and 6 - M F G caused alteration in the staining characteristics o f the myelin, which b e c a m e swollen, vacuolated, thinned and displaced f r o m the S c h w a n n sheath. Higher concentrations produced additional c h a n g e s , w i t h v a r i a t i o n s w i t h e a c h d r u g . HtN2 caused marked axis cylinder swelling and eventual fragmentation as well as intrafascicular hemorrhage and edema. A139 and V L B also caused axis cylinder fragmentation, but they also produced an unusual intrafascic ular deposition of hyaline material rarely associated with hemorrhage. The myelin and axis cylinder changes with 6-~/LIDG w e r e less remarkable. Vasa n e r v o r u m histologically appeared normal. Corresponding nerve conduction changes w e r e noted with HN2, A139, VLB and 6-MI~ but not with Cytoxan or controls. The anticancer agents that produced the most remarkable changes w h e n applied locally caused relatively benign histologic changes following regional leg perfusion. E a c h drug was dissolved in the entire perfusate prior to onset of perfusion. It has subsequently been found that H N 2 loses its activity rapidly in vitro in blood o r p l a s m a 14 and that H N 2 and A 1 3 9 are rapidly cleared f r o m the blood in vivo. 13.14 The lack of remarkable nerve changes following perfusion with these dosages was felt to be due to in vitro deterioration of drug in the perfusate and to rapid uptake of drug by all tissues of the perfused leg. T o investigate the first of these two possibilities, deterioration of drug in vitro in the perfusate was reduced by injecting H N 2 directly into the arterial inflow catheter after perfusion was established (Table 8). This Table 8. Histolagic Changes in the Sciatic Nerve of the Dog Five Days Following HN2 Injection in the Arterial Inflow Catheter During Regional Lower Limb Perfuslon for 20 Minutes
Dose (mg./kg.) 1.0 2.5 5.0
HistologicNerve Changes Demye[ini zation Demyelinization; axis cylinder fragmentation; epineurial thickening Demyelinization; axis cylinder fragmentation; epineurial thickening
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F i g u r e 6. C r o s s sections of n e r v e s taken f r o m legs p e r f u s e d withHN2 d i s s o l v e d throughout the p e r f u s a t e or injected in the a r t e r i a l inflow c a t h e t e r , x 160. A, L F B - P A S stain, 5.0 rag. p e r kg. d i s s o l v e d in p e r f u s a t e , showing v e r y l i t t l e a l t e r a t i o n of r o s e t t e p a t t e r n of myelin. B, Bodian stain, 3.0 mg. per kg. d i s s o l v e d in p e r f u s a t e , showing r e l a t i v e l y n o r m a l axis c y l i n d e r s . C, L F B - P A S ' s t a i n , 2.5 rag. per kg. injected in a r t e r i a l inflow c a t h e t e r , resulting in s e v e r e loss o s D, Bodian stain, 2.5 rag. per kg. injected in a r t e r i a l inflow c a t h e t e r , showing i n t r a f a s c i c u l a r edema and loss os axis c y l i n d e r s . t e c h n i q u e of H N 2 a d m i n i s t r a t i o n r e s u l t e d in m o r e s t r i k i n g p a t h o l o g i c c h a n g e s than had b e e n s e e n w h e n HN2 w a s d i s s o l v e d t h r o u g h o u t the p e r f u s a t e ( F i g . 6). T o i n v e s t i g a t e t h e s e c o n d p o s s i b i l i t y , c h l o r o e t h y 1 - C 14 l a b e l e d HN2 w a s p e r f u s e d to t h e l e g s o f two d o g s . T h i r t y m i n u t e s following p e r f u s i o n , the s c i a t i c n e r v e , f e m o r a l a r t e r y , f e m o r a l v e i n and a p o r t i o n of m u s c l e f r o m t h e p e r f u s e d a n d f r o m the o p p o s i t e l e g s w e r e e x a m i n e d for the p r e s e n c e o f r a d i o a c t i v i t y ( T a b l e 9). T h e t i s s u e s from the perfused leg had the highest concent r a t i o n s a s c o m p a r e d to t h o s e o f t h e t o t a l b o d y , Table 9. Counts Per Minute (CPM) of C 14 in Tissue
Samples 30 Minutes After Regional Lower Limb Perfusion with 15/~c. of HN2- C 14
Tissue Samples Femoral artery Femoral vein Muscle Sciatic nerve
CPM/gm. Perfused leg Opposite/eg 1554 647
51 0
62 13
9 0
b u t t h e c o n c e n t r a t i o n in t h e n e r v e w a s l o w e s t . T h u s , it w o u l d s e e m t h a t b o t h in v i t r o d r u g d e t e r i o r a t i o n in the p e r f u s a t e and uptake of d r u g by s e v e r a l t i s s u e s o t h e r t h a n n e r v e o c c u r r e d in t h e e x p e r i m e n t a l l i m b p e r f u s i o n s ( T a b l e 3). Q u a n t i t a t i v e l y , n e r v e t i s s u e d i d not selectively, concentrate HN2, when compared with muscle, following experimental regional limb perfusion. Therefore the pcripheral neuropathies seen following perf u s i o n s in p a t i e n t s probably represent a q u a l i t a t i v e t i s s u e s u s c e p t i b i l i t y in t h e p r e s ence of a relatively high local concent r a t i o n of the a n t i t u m o r substance, while neuropathies following intravenous drug administration may also reflect this peculiar sensitivity. Thes e studies indicate a primary alteration in m y e l i n s t r u c t u r e , a s s o c i a t e d w i t h a n e r v e c o n d u c t i o n d e f e c t . The s u s c e p t i b i l i t y of m y e l i n m a y be d e p e n d e n t upon the lipid s o l u b i l i t y of HN2, which probably enhances its entrance into cells, l's'? The hemorrhage and edema of the i n t r a f a s c i c u l a r spaces characteristic of h i g h e r d o s a g e s a r e i d e n t i c a l with the
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F i g u r e 7. Sciatic n e r v e of patient six days following pelvic perfusion with 60 rag. HN2. A, Longitudinal section, Bodian stain, showing loss of axis c y l i n d e r s , x 160. B, C r o s s section, L F B - P A S stain, showing pale staining of m]~elinand d i s p l a c e m e n t a w a y f r o m Schwann sheath, x 160. C, C r o s s section, L F B - P A S stain, showing subepineurial h e m o r r h a g e , x 64. vascular necrosis described in earlier s t u d i e s 4 and in the b r a i n following c a r o t i d a r t e r y i n j e c t i o n . S, 9 T h e s a m e c o n c e p t s w o u l d a p p e a r valid for the r e m a i n i n g a c t i v e a n t i tumor systems that were studied. The postperfusion peripheral neuropathy of a p a t i e n t h a s b e e n s t u d i e d a t a u t o p s y . P e l v i c p e r f u s i o n w a s d o n e w i t h 60 rag. of HNZ i n j e c t e d d i r e c t l y i n t o t h e a r t e r i a l i n f l o w catheter. Bilateral sciatic nerve paralysis d e v e l o p e d on the day following p e r f u s i o n , five days before death. Demyelinization, axis cylinder fragmentation, edema and focal hemo r r h a g e was found in the s c i a t i c n e r v e s {Fig. 7). These observations do n o t e x p l a i n the m a r k e d r e l i e f of p a i n t h a t f o l l o w s p e r f u s i o n of m a l i g n a n t t u m o r s w i t h c o m m o n l y u s e d a l k y l a t i n g a g e n t s i n the l a c k of a n y o b v i o u s neurologic changes, More sophisticated nerve fiber conduction studies might elucidate this p h e n o m e n o n . One p a t i e n t with tic d o u l o u r e u x and bundle-branch heart block has been perf u s e d t h r o u g h the e x t e r n a l c a r o t i d a r t e r y - i n t e r n a l j u g u l a r v e i n w i t h A 1 3 9 . R e l i e f of tic p a i n has b e e n o b t a i n e d for nine m o n t h s with
concomitant but mild radiomimetic skin c h a n g e s a n d s l i g h t l o s s of p a i n d i s c r i m i n a tion. Adequate dosage titration may permit an e x t e n s i o n of this p r o v o c a t i v e t e c h n i q u e of pain control. SUMMARY S t u d i e s a r e d e s c r i b e d w h e r e i n the s c i a t i c n e r v e of the dog w a s e x p o s e d to c h e m o t h e r a p e u t i c a g e n t s by t o p i c a l a p p l i c a t i o n , s u b e p i n e u r i a l i n j e c t i o n o r r e g i o n a l l i m b pe.rfusion. Histologic and nerve potential conduction changes are reported. The relationof t h e s e f i n d i n g s to v a r i o u s neuropathies observed clinically following treatment with t h e s e a g e n t s is d i s c u s s e d . REFERENCES I. Boyland, E.: The toxicity of a l k y l - b i s ({~chloroethyl) a m i n e s and of the products of their reaction with water. Brit. J. Pharmacol., l: Z47, 1946. 2. Conrad, M. E., J r . , and Crosby, W. H.: k{assive nitrogen m u s t a r d therapy in Hodgkin's disease with protection of bone m a r r o w by tourniquets. Blood, 16: 1089, 1960.
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3. C r e e c h , O. J . , Ryan, i%. F . , and K r e m e n t z , E. T.: R e g i o n a l c h e m o t h e r a p y by i s o l a t e d p e r f u s i o n in t h e t r e a t m e n t o f m e l a n o m a of the e x t r e m i t i e s . P l a s t . & R e c o n s t r u c t . S u r g . , 28: 333, 1961. 4. D a n i e l l i , J . F . : C e l l P h y s i o l o g y and P h a r m a c o l o g y , New York, E l s e v i e r P u b l i s h i n g Co., Inc., 1950. 5. F e l l , H. B., and A l l s o p p , C. B.: T i s s u e c u l t u r e e x p e r i m e n t s on the b i o l o g i c a l a c t i o n o f m e t h y l b i s ( ~ - c h l o r o e t h y l ) a m i n e and its h y d r o l y s i s p r o d u c t s . C a n c e r Ftes., 9: 238, 1949. 6. F r e n c h , J . D., W e s t , P. M., v o n A m e r o n g e n , F . K., and M a g o u n , H. W.: E f f e c t s of i n t r a c a r o t i d a d m i n i s t r a t i o n of n i t r o g e n m u s t a r d on n o r m a l b r a i n and b r a i n t u m o r s . J . N e u r o s u r g . , 9: 378, 1952. 7. G i l m a n , A., a n d P h i l l i p s , F . S.: T h e b i o l o g i c a l a c t i o n s and t h e r a p e u t i c a p p l i c a t i o n o f f i - c h l o r o e t h y l a m i n e s and s u l f i d e s . S c i e n c e , 103:409, 1946. 8. H e r t z , R.: T h e r a p e u t i c t r i a l s with v i n c a l e u k o b l a s t i n e in p a t i e n t s with M e t h o t r e x a t e - r c s i s t a n t cho r i o c a rc i n o m a . C a n a d i a n Cane e r C o n f e r e n c e, N e w York, A c a d e m i c P r e s s , 1961, p. 383. 9. K1opp, C. T., A1ford, T. S., B a t e m a n , J., B e r r y , G. N., and W i n s h i p , T.: F r a c t i o n a t e d intra-arterial cancer c h e m o t h e r a p y with m e t h y l b i s (fl- chlo r o e t h y l ) a m i n e h y d r o c h l o r i d e . Ann. S u r g . , 132: 811, 1950. 10. Lawrence, W., Jr., Kuehn, P., Masle, E. T., and Miller, D. G.: A n abdominal tourniquet for regional chemotherapy. J. Surg. Res., I: 142, 1961. 11. L y o n s , W. R., and Woodhall, B.: A t l a s of P e r i p h e r a l N e r v e I n j u r i e s . P h i l a d e l p h i a , W. B. S a u n d e r s C o . , 1949. lZ. M a h a l e y , I%{. S., J r . , H u n e y c u t t , H., Boone, S., and Woodhall, B.: L o c a l i z a t i o n of m e t h y l bis (2-chloroethyl-1, 2-C14)amine hydrochloride in n e r v o u s t i s s u e a f t e r i n t r a v e n o u s i n j e c t i o n o r r e g i o n a l c e r e b r a l p e r f u s i o n in d o g s . C a n c e r C h e m o t h e r . R e p . , 11:29, 1961. 13. M a h a l e y , M. S., J r . , M a h a l e y , J a n e , and Woodhall, B . : B i o a s s a y o f p l a s m a l e v e l s o f A 1 3 9 and m a n n i t o l m u s t a r d f o r u s e in d e t e r m i n i n g blood l e v e l s d u r i n g r e g i o n a l b r a i n c a n c e r p e r f u s i o n s . C a n c e r C h e m o t h e r . R e p . , 11: 33, 1961.
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M a h a l e y , M. S., J r . , and WoodhaI1, 13.: P l a s m a l e v e l s of a l k y l a t i n g a g e n t s d u r i n g r e g i o n a l c h e m o t h e r a p e u t i c p e r f u s i o n s . J. Surg. Res., 1: 285, 1961.
15.
M a i m , J. Ft., H e r t e r , F . P., F r i c k , H. C., Long, M. E., and D e m e t z , A.: E v a l u a t i o n o f i s o l a t e d p e r f u s i o n c h e m o t h e r a p y in the t r e a t m e n t of a d v a n c e d m a l i g n a n t d i s e a s e . S u r g . F o r u m , I0:743, 1959.
16. Shingleton, W. W., Parker, R.T.,andMahaley, S.: Abdominal perfusion for cancer chemotherapy. Ann. Surg., 152: 583, 1960. 17. Sholes, D. ~I.: Pelvic perfusion with nitrogen mustard for cancer: a neurological complication. A m . J. Obst. & Gynec., 80:481, 1960. 18. Strelinger, A. L., Gilbert, L., and Shoshkes, M.: Extracorporeal regional perfusion with nitrogen mustard for the palliation of amalignant tumor. J. N e w a r k Beth Israel Hosp., II: 79, 1960. 19. Vaitkevicius, V.K., Talley, R. W., and Brennan, M. J.: Cytological and clinical observations during vincaleukoblastine therapy of disseminated cancer. Proc. A m . A. Cancer Ftes., 3: 275, 1961. 20. %Vill, J. J., Korst, D. 1%., Monto, R. W., and Hurley, J. D.: Phase II evaluation of vincaleukoblastine. A study by the MidwestCooperative Chemotherapy Group. Proc. A m . A. Cancer Res., 3:Z78, 1961. 21. Woodhall, B., Hall, K., Mahaley, S., Jr., and Jackson, J.: Chemotherapy of brain cancer: experimental and clinical studies in localized hypothermic cerebral perfusion. Ann. Surg., 150: 640, 1959. 22. Woodhall, B., Pickrell, K. L., Georgiade, N. G., Mahaley, M. S., Jr., and Dukes, H. T.: Effect of hyperthermia upon cancer chemotherapy; alSplication to external cancers of head and face structures. Ann. Surg., 151: 750, 1960. 23. Young, W. G., Jr., Lesage, A. M., Dillon, M. L., Lee, J. M., Callaway, H. A., Jr., and Reeves, J. %V.: Chemotherapy of intrathoracic neoplasms employing differential pelvic perfusion hypotherrnia. Ann. Surg., 154: 372, 1961.