Acta neuropath. (BEE.) 24, 244--255 (1973) 9 by Springer~Verlag 1973

Distribution of Tetanus Toxin in Rat Peripheral Nerve Trunks during the Development of Local Tetanus L l o y d E. King, Jr., a n d A l e x a n d e r A. F e d i n e c Veteran's Administration Hospital, Electron Microscope Research Unit, Memphis, Tennessee, U.S.A., and Departments of Anatomy and Medicine, University of Tennessee Medical Units, Memphis, Tennessee 38103, U.S.A. Received November 6, i972

Summary. Mouse bioassay studies were made on the tetanus toxin content of three different segments of both sciatic nerve trunks after unilateral gastrocnemius muscle injections. Multiple toxin dosages, sampling times, nerve transections, and nerve dissections to produce three samples per segment (undissected nerve, epineurium, and perineurium plus endoneurium) were used. The results showed that after i.m. injections a dosage dependent bilateral toxin distribution could be detected in the undissected nerve or its epineurium. The characteristics of this epineurial toxin distribution are discussed. A unilateral toxin distribution was detected only in the "stripped" (perineurinm plus endoneurium) nerve segment from the injected extremity. This unilateral toxin distribution had the characteristics of centripetal toxin transport along the tissue spaces or axons to the spinal cord. This unilateral centripetal toxin gradient was temporally related to the onset of local tetanus. Both the toxin gradient pattern and local tetanus due to i.m. injected toxin could be prevented by nerve transection. These data suggest that peripheral nerves do transport tetanus toxin to the spinal cord and that this transport is an important factor in the pathogenesis of local tetanus. Key words: Tetanus Toxin -- Bioassay -- Local Tetanus -- Peripheral Nerve Transport.

Introduction U n d e r m o s t e x p e r i m e n t a l conditions, a b i l a t e r a l d i s t r i b u t i o n of t e t a n u s t o x i n has been n o t e d in homologous p e r i p h e r a l nerves following u n i l a t e r a l t o x i n inject i o n into t h e corresponding muscle (Laurence a n d W e b s t e r ; L a m a n n a a n d Carr; K r y z h a n o v s k y i i , 1967b). This b i l a t e r a l t o x i n d i s t r i b u t i o n is t h e basis for criticisms of t h e N e u r o n a l T o x i n T r a n s p o r t T h e o r y of Marie a n d Morax. This t h e o r y proposes t h a t t e t a n u s t o x i n is t r a n s p o r t e d c e n t r i p e t a l l y via t h e p e r i p h e r a l nerves f r o m t h e site of infection or e x p e r i m e n t a l t o x i n injection to t h e spinal cord to p r o d u c e local (unilateral) t e t a n u s . K r y z h a n o v s k y i i ( 1 9 6 7 a ) i n j e c t e d i.m. v e r y low t o x i n dosages into m a n y species a n d d e t e c t e d a u n i l a t e r a l t o x i n d i s t r i b u t i o n w i t h i n p a s t e e x t r a c t s of t h e corresponding u n d i s s e c t e d p e r i p h e r a l nerve segments. These studies d i d n o t d e t e r m i n e w h a t a n a t o m i c a l c o m p o n e n t of t h e p e r i p h e r a l nerve t r u n k s c o n t a i n e d t o x i n nor w h y o n l y low t o x i n dosages p r o d u c e d such a n effect. I n view of c u r r e n t i n t e r e s t in the t r a n s p o r t of various substances b y p e r i p h e r a l nerves ( H a b c r m a n n ; L a s e k ; K a r l s s o n a n d S j o s t r a n d ; Ochs et al.), t e t a n u s t o x i n t r a n s p o r t a p p e a r e d to be a p r o m i s i n g i n v e s t i g a t i v e area.

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T h e bioassay m e t h o d of t o x i n localization has been e x t r e m e l y useful despite the supposed l i m i t a t i o n s of s e n s i t i v i t y a n d of localization. Th e bioassay specifically detects t h e d i s t r i b u t i o n of a c t i v e t e t a n u s t o x i n molecules, whereas t h e other localization m e t h o d s do not. I t is reasonable to assume t h a t t h e d i s t r i b u t i o n of an a c t i v e t r a c e r labeled t e t a n u s t o x i n molecule or its labeled f r a g m e n t (all, lslI, ~25I, fluoresceinisothiocyanate, ferritin, or horseradish peroxidase) m a y n o t be t h e same in rive as t h a t of a c t i v e u n l a b e l e d t o x i n molecules. Immunohistolo~ gic~l m e t h o d s m a y localize a c t i v e as well as i n a c t i v e antigenic f r a g m e n t s of the t o x i n molecule (Fedinee). P r e v i o u s l y (King), it was f o u n d possible to separate by mierodissection methods, t h e e p i n e u r i n m f r o m th e p e r i n e u r i u m an d e n d o n e u r i n m of r~t sciatic nerves. These n e r v e c o m p o n e n t s were used in a mouse bioassay m e t h o d to d e t e r m i n e the t e t a n u s t o x i n c o n t e n t of t h e ipsilateral a n d contral~teral sciatic n e r v e t r u n k at diffel~ent times after i.m. t o x i n injections. These bioassay m e t h o d s were used to clarify th e role of t h e peripheral n er v e c o m p o n e n t s in t h e u p t a k e and distribution of i.m. i n j e c t e d t o x i n during the d e v e l o p m e n t of local tetanus.

Materials and Methods 800 adult Albino male rats of Wistar strain weighing between 280 znd 300 g and 3620 male C57 B1/6J black mice weighing between 20 and 25 g obtained from ore' central animal quarters were used in the series of experiments. The animals had been kept on a diet of commercial pellets and water ad libidum. Temperature and space allowed for movement were kept uniform. All animals were observed at 30 rain intervals for the first 24 h following injection and then at 6 h intervals for the duration of the experiment. Pentobarbital sodium (Diabutal| 50 mg/kg, injected intraperitoneally (i.p.) was used for surgical anesthesia. The animals were sacrificed with an overdose of the anesthetic. Purified tetanus toxin and antitoxin were kindly provided by William C. Latham of The Institute of Laboratories, Massachusetts Department of Public Health, Boston, Massachu serfs (Latham et el.). The purified toxin (Batch CPTxn-21-AS-11) contained 8• 107 mouse minimum lethal doses (MLDs) and 1000 I.U. Lf doses/ml. The toxin was lyophilized, reconstituted with sterile saline, and toxicity reconfirmed immediately prior to use in the experiments. The direct method of graded dilutions was used to determine the minimum dose of the lyophilized toxin which would kill 100~ of the intraperitoneally injected mice in 144 h, one minimum lethal dose (Imbriano). The MLD following i.p. injection was found to be 0.05 ml/100 g body weight of a 1 : 1 million dilution of the ]yophilized toxin (2 • 107 mouse MLDs/ml). A dosage of 100; 600; 1000; 2500; 6000 MLDs/100 g body weight/0.05 ml was selected as the i.m. or i.v. dosage for rats on the basis of these preliminary experiments. The equine antitoxin (Lot No. LA 136) had a potency of 380 Lf/ml. The antitoxin, serially diluted with sterile saline, was injected intravenously into rats, An antitoxin dosage of 10 I.U./0.05 m]/100 g body weight prevented the development of symptoms in rats injected i.m, with 600MLDs/100g body weight. Sterile 1 ml disposable syringes (Sty}ex| fitted with a 23 gauge needle were used for all iicjections except for the nerve segments which required the larger I9 gauge needle. For the bioassay of tetanus toxin content of rat tissues, the tissue samples were injected (i.p.) into mice and the developing symptoms of tetanus recorded daily. Mice dying of tetanus within 144 h following injection (i.p.) were considered to have received at least one MLD of toxin. A five point scoring system was devised to evaluate the sublethal tetanus symptoms in mice. The points were awarded on the following basis: 1 point--clonie facial spasm; 2 points--persistent trismus; 3 points--lordosis; 4 points--generalized tetanus; 5 points-tetanus death. A cumulative score was recorded for each group of i.p. injected mice for each

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day of the experimental period. The cumulative score was converted into ~ Tetanus Effect (~ T.E.) b y the following formula. Cumulative score (0--5 points/mouse) • 100. Maximum score (5 • mice/group)

Tetanus Toxin Toxicity, Speci/icity, and Stability I. Preliminary Experiments Group a. To test the specificity of the tetanus toxin used, 10 anesthetized rats were prepared b y exposing b o t h femoral veins in the femoral triangle. The left femoral vein was injected with 600 mice MLDs/100 g body weight of tetanus toxin. Immediately, the right femoral vein was injected with 10 I.U./100g body weight of equine antitoxin. The incisions were sutured and the rats allowed to recover before placing t h e m into cages for observations. Group b. The reaction of 40 B1/6J mice to rats' tissues containing no toxin was tested. The surgical procedures, the tissue samples obtained, and the mouse bioassay methods used represent the basic experimental methods and will be described in detail only for this group. Any exceptions to those basic methods will be described in the appropriate groups. R a t venous blood samples were obtained from the femoral veins a n d injected in 0.1--1.0 cc volumes intraperiteneally (i.p.) into the bioassay mice. The r a t sciatic nerves were exposed on the lateral aspect of the rat's thigh from its formation a t the lumbar plexus to its division into the tibial a n d peroneal nerves. The total length of the sciatic nerve removed was approximately 6 cm. The 6 cm sciatic nerve t r u n k was t h e n divided into a n upper, middle, and lower segment each 2 cm long. Using 0000 silk ligatures, each 2 cm segment was placed onto a Parafilm | strip in a labeled container. The container was sitting on ice to reduce nerve segment dessication prior to the microscopic dissection. U n d e r the dissecting microscope the nerve segment could be stripped easily of its loose epineurial connective tissue. I f the epineurium was removed from the particular sciatic nerve segment, t h e n t h a t nerve segment was designated as a "stripped" nerve segment. After removal, all "stripped" sciatic nerve segments (perineurium plus endoneurium) were t h e n weighed on a Mettler balance to insure uniformity of sample size. Whole nerve segments or "stripped" nerve segments t h e n were placed into appropriately labeled, individual, saline filled syringes for bioassay injections. The injection of the nerve segments was facilitated b y cutting the nerve segment into fragments just prior to placing in the syringes. The mice injected with the rat tissue components were placed into labeled cages a n d observed daily for 14 days. A t the end of the observation period, the mice were sacrificed a n d examined at necropsy for any sublethal effects of the r a t tissues on the mice. Group c. The in vitro stability and affinity of tetanus toxin for rat's blood and sciatic nerve segments was also tested. Ten control rats not injected with toxin were anesthetized. The following r a t tissue samples were obtained from these ten rats with the same methods described above: 1.0.5 cc of the rat's blood, 2. right lower "stripped" nerve segment, 3. the epincurium from the same segment, and 4. the undissected left lower sciatic nerve segment. Six groups of ten one ec syringes were filled with 0.5 cc of sterile saline-toxin containing ten mouse MLDs. Before the r a t ' s blood clotted, the blood was added a n d mixed thoroughly with the saline-toxin. The r a t sciatic nerve tissues were t h e n p u t into appropriately labeled syringes b y adding 0.5 ce of sterile saline to suspend the "stripped" nerve segments or its epineurial tissue components. To the t w e n t y syringes containing only the saline-texin, another 0.5 cc of sterile saline was added to give a final volume of one cc. Ten of these syringes were t h e n used to inject mice with the unincubated saline-toxin. The death of these mice would be used to confirm the lethality of the added saline-toxin and to serve as toxin controls for the remaining five groups of toxin-containing syringes. The remaining fifty syringes were t h e n incubated for one hour at 37~ After this incubation period, the contents of each syringe were t h e n injected i.p. into a mouse. The mice were placed in labeled cages for observations. The symptoms and time of d e a t h were recorded for each mouse from the six groups. Group d. To determine the onset of local tetanus, bloodborne tetanus, a n d the time of r a t d e a t h after toxin injections the following procedures were used: :Five groups of ten rats each

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were injected with toxin in the left gastrocnemius muscle to produce local tetanus. The five groups of rats were based on the toxin dosage used: 1. 100, 2. 600, 3. 1000, 4. 2500, or 5. 6000 mouse MLDs per 100 g rat's body weight. Similarly, to determine the onset of bloodborne tetanus a n d the time of rat d e a t h after intravenous toxin injections the following procedures were used: Five groups of five rats each were injected with toxin into the right femoral vein to produce bloodborne tetanus. These five groups were based upon the five toxin dosages used above for the intramuscular toxin injections. The time of the development of tetanus symptoms and time of death was recorded.

I I , Experimental Series Group a. To determine if removal of different segments of the sciatic nerve would alter toxin uptake of the remaining nerve segments, the following procedures were used: The lower 2 em of the sciatic nerve was transected a n d removed from the left lower extremity of 28 rats. Two weeks later 14 rats were injected with 2500 mouse MLDs and 14 rats with the 6000 MLDs of tetanus toxin into the left gastrocnemius muscle. Two rats from each dosage group were selected as toxicity controls to be observed for local tetanus, bloodborne tetanus, a n d death. The remaining rats (12 rats per dosage level) were observed for signs of spastieity until sacrificed. From each dosage level, 6 rats were anesthetized at 18 h and 6 rats at 24 h after toxin injection. Two cc's of venous blood was withdrawn prior to surgical exposure, ligation, and removal of the remaining left sciatic nerve segments (middle 2 cm and upper 2 cm) and the entire right sciatic nerve (6 cm). Three of the 6 experimental rats from each dosage group (2500 and 6000 MLD/100 g B.W.) and each sacrifice time (18 and 24 h after injection) were used to obtain undisseeted nerve segments for toxin bioassay. The remaining 3 rats from each dosage group a n d each sacrifice time were sacrificed and their sciatic nerves were dissected into their epineurial and "stripped" nerve components for toxin bioassay. Group b. To determine if toxin would accumulate ill the lower sciatic nerve segment nearest the toxin injected left gastrocnemius muscle, the following experiment was performed. The middle sciatic nerve segment was transected and removed from the left lower extremity of 28 rats 2 weeks prior to toxin injections into the left gastrocnemius muscle. W i t h this exception, the toxin dosage, the time of sacrifice, and the n u m b e r of animals used were the same as t h a t described above. Group c. The contribution of the t o x i n located within t h e intraneural blood vessels to the total toxicity of the r a t sciatic nerve segments was studied b y using i.v. injections of different toxin dosages in the following manner: After anesthesia, i.v. toxin injections of 100, 600, 1000, 2500 or 6000 mouse MLDs per 100 g rat's B.W. were made into the right femoral vein of 5 groups of 14 rats each. Two control rats from each toxin dosage group were placed in separate cages for continual observation of the symptoms and time of death after the i.v. injection of the various toxin dosages. Six rats from each dosage group were used to obtain undissected sciatic nerve segments. The remaining 6 rats from each dosage group were used to obtain epineurial a n d "stripped" nerve components of homologous nerve segments for comparison of the t o x i n contents of t h e segments. Five minutes after toxin injections into the rats' left femoral vein, b o t h sciatic nerves were ligated a n d removed. After nerve ligation, venous blood samples were collected and injected i.p. into mice for bioassay. The undissected nerve segments from 6 rats from each t o x i n dosage group were t h e n divided into upper, middle and lower nerve segments a n d injected into mice for bioassay. The epineurium and the "stripped" nerve segments from the remaining 6 rats from each toxin dosage group were also divided into upper, middle, and lower segments a n d injected for mouse bioassay. To determine if sampling time was critical to the a m o u n t a n d distribution of i.v. injected toxin the experiments were repeated using a sacrifice time of 12, 18, 22 and 28 rain after the i.v. toxin injections. The toxin dosages, grouping, and samples were identical to the 5 rain sacrifice experiments. Group d. To determine the presence a n d location of tetanus toxin within the different segments of the entire rat sciatic nerve and its connective tissue components after i.m. toxin injections, the following methods were used. Five groups of 110 rats each were injected in their left gastrocnemius muscle with one of t h e following dosages: 100, 600, 1000, 2500 or 6000 mouse MLDs per 100 g B.W. All rats from each dosage group were frequently observed

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for the development of local or bloodborne tetanus until they were sacrificed or died of tetanus. Twenty rats from each dosage group were anesthetized and sacrificed at 1, 6, 12, 18, or 24 h after the i.m. toxin injection. The remaining 10 rats from each dosage group served as tetanus controls and were observed for the progressive development of local or bloodborne tetanus, or death. One cc of rat's blood and the upper, middle, and lower nerve segments were obtained from both extremities of all rats by the methods described in Group Ib. From each group of 20 rats half of their upper, middle and lower nerve segments were injected without microdissection. The remaining sciatic nerve segments were dissected into epineurial and "stripped" nerve segments for comparison with homologous nerve segments. The methods of injection, sampling, labeling, bioassay, and observation were the same as described in Group I b.

Results

I. Tetanus Toxin Toxicity, Specificity, and Stability Group a. All r a t s i n j e c t e d i.v. w i t h 600 m i c e M L D s / 1 0 0 g B . W . o f p u r i f i e d t e t a n u s t o x i n s m w i v e d w i t h o u t s h o w i n g a n y t e t a n u s s y m p t o m s i f 10 I . U . / 1 0 0 g B . W . of a n t i t o x i n was g i v e n i.v. i m m e d i a t e l y a f t e r t h e t o x i n i n j e c t i o n . Group b. T h e a c c u r a c y of t h e m i c r o d i s s e c t i o n was c o n f i r m e d b y H e m a t o x y l i n and Eosin stains of histological sections of randomly

selected rats' sciatic nerve

c o m p o n e n t s . N o a d v e r s e r e a c t i o n s w e r e n o t e d in m i c e i n j e c t e d i.p. w i t h saline, r a t ' s blood, or s a m p l e s of t h e r a t sciatic n e r v e . Group c. I n c u b a t i o n of r a t sciatic n e r v e s e g m e n t s , " s t r i p p e d " n e r v e or e p i n e u r i u m w i t h t e t a n u s t o x i n for 1 h a t 3 7 ~ s l i g h t l y p o t e n t i a t e d or p r o l o n g e d t h e a c t i v i t y of t h e a d d e d t e t a n u s t o x i n as c o m p a r e d w i t h t h e t e t a n u s t o x i n saline c o n t r o l s i n c u b a t e d u n d e r t h e s a m e c o n d i t i o n s . Mice i n j e c t e d w i t h t h e n e r v e t i s s u e - t e t a n u s t o x i n c o m b i n a t i o n s d i e d 18 to 26 h earlier t h a n t h o s e m i c e i n j e c t e d w i t h s a l i n e - t o x i n c o m b i n a t i o n ( 9 2 - - 9 6 h). Group d. T h e o n s e t of local t e t a n u s , b l o o d b o r n e t e t a n u s , a n d t h e t i m e of r a t d e a t h was i n v e r s e l y p r o p o r t i o n a l to t h e t o x i n dosage. I n t r a v e n o u s t o x i n i n j e c t i o n s d i d n o t p r o d u c e a n y local t e t a n u s s y m p t o m s . T h e o n s e t of g e n e r a l i z e d t e t a n u s a n d t e t a n u s d e a t h was n o t d e p e n d e n t u p o n t h e r o u t e of i n j e c t i o n w i t h b o t h r o u t e s p r o d u c i n g s i m i l a r p a t t e r n s (Table 1). Table 1. Onset of symptoms and time of death of rats after the i.m. or i.v. injection of

tetanus toxin Toxin dosage a

Local tetanus i.m. b i.v. c

Generalized tetanus i.m. i.v.

Death fromtetanns i.m. i.v.

100 600 1000 2500 6000

36--40 24--28 22--24 20--22 18--20

Hours a 88--100 44-- 48 36-- 42 28-- 32 23-- 27

120--144 68-- 72 60-- 66 44-- 48 32-- 36

a b c a to be e

_ e -----

88--100 44-- 48 36-- 42 28-- 32 23-- 27

120--144 68-- 72 60-- 66 44-- 48 32-- 36

The toxin dosage is expressed as mouse MLDs per 100 g rat's body weight. The intramuscular toxin injections were made into the rat's left gastrocnemius muscle. The intravenous toxin injections were made into the rat's right femoral vein. The number of hours after the toxin injection when the specified symptoms were noted persistent and reproducible. Local tetanus does not appear after intravenous toxin injections.

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I I . Experimental Series Group a. The removal of either the lower or middle sciatic nerve segment prevented the development of local tetanus in the toxin injected extremity. The nerve transection did not prevent the development of generalized tetanus or death unless specific antitoxin was injected i.v. to neutralize toxin absorbed after the i.m. toxin injections. The experimental rats used in this group were sacrificed at or prior to the expected development of generalized tetanus and were not injected with specific antitoxin. If the lower sciatic segment was removed from the toxin injected extremity, no toxin could be detected in the remaining "stripped" nerve segments from that extremity (middle and upper) or in the three "stripped" segments from the right sciatic nerve. A lethal dose of toxin was detected in the epineurium from these segments and the rats' blood at both sacrifice times (]8 and 24 h) and toxin dosages (2500 and 6000 mouse MLDs/ 100 g B.W.) used.

Group b. If the middle segment was removed from the toxin injected extremity, no toxin could be detected in the three "stripped" segments from the right sciatic nerve or the upper "stripped" nerve segment from the injected extremity. Toxin was detected in the epineurinm from these segments and in the rats' blood at both sacrifice times with either toxin dosages. In the injected extremity, the undisseeted lower nerve segments, its "stripped" nerve component (perineurium plus endoneurium) and its perineurinm were uniformly lethal (100~ T.E.) for the bioassay mice at either sampling time or dosage. Group c. All control rats developed generalized tetanus and died at times proportional to the toxin dosages (Table 1). With the dosages used, 0.1--0.5 ee of the rats' blood samples were always lethal for the bioassay mice. When the time of rat sacrifice was 5 rain after the i.v. injection of any toxin dosage used, no toxin was detectable in any rat's "stripped" sciatic nerve segment. At this time, toxin was detectable in the blood and in the epineurium of the same segments. However, rats sacrificed 18 to 30 min after the i.v. injection of the 6000 mouse MLD dosage were noted to have detectable toxin not only in the blood and epineurium, but also within their "stripped" nerve segments. All 2 em segments from both extremities produced similar toxicity in the bioassay mice. Group el. Tetanus toxin was detectable in 0.1 ml of rats' blood from 1 to 24 h after the i.m. injection of any dosage used. The distribution within the different samples was dependent upon the toxin dosage, the sampling time, the anatomical position of the nerve segment, and the specific nerve component used for hieassay. The sciatic nerve toxin distribution pattern of the two extremities only differed markedly with respect to the toxin content of the lower nerve segments and the "stripped" nerve samples. These two special eases will be considered in detail below. With these two exceptions the following similarities were noted in the overall toxin distribution pattern in the two extremities: 1. the undisseeted nerve sample and its homologous epineurial sample were very similar in toxin content at each various toxin dosage and sampling time; 2. in both extremities these two samples usually reached a plateau of toxicity at 12 h after injection; 3. the pattern of these two samples from all three right nerve segments was the same as that of comparable samples from the left middle and upper nerve

L.E. King, Jr., and A. A. Fedinec:

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Fig. 1. Tetanus toxin content of left sciatic nerve segments after i.m. injection of 100, 600, 1000, 2500, or 6000 MLDs/100 g body weight

segments (Fig.l, middle upper, A and B). I f the "stripped" nerve values are omitted from Fig. 1 upper, A and B, then the pattern of all three right sciatic nerves is readily visualized. By comparing these values from the entire right sciatic nerve with those obtained from the left lower segment, the following differences were noted: 1. the undissected nerve and its homologous epineurial sample from the left lower segment were not as similar as those of all other segments from both extremities; 2. in contrast to the steadily increasing toxin content of all other segments, the toxin content of the left lower segment was initially very high with a subsequent decline in toxicity at the 100 to 600 MD dosage range; 3. in contrast to all other segments, increasing the toxin dosage above the 600 MD dosage range; 3. in contrast to all other segments, increasing

Tetanus Toxin in P~at Peripheral Nerve

25t

the toxin dosage above the 600 MLD level produced a maximal tetanus effect (100~ in all nerve components (undissected, epineurial and "stripped" nerve) from the left lower segment throughout the 24 h of sampling. The amount of toxin the undissected "stripped" nerve contributes to the total toxicity of the left lower nerve segment is then obscured when toxin dosages above 600 MLD were used. The toxin distribution pattern in the left "stripped" sciatic nerve is obtained by comparing the three left segments (lower, middle and u p p e r ) w i t h each other. The "stripped" nerve segment from the entire right sciatic nerve did not contain any detectable toxin under these experimental conditions. The unilateral "stripped" nerve toxin pattern in the left sciatic nerve had the following characteristics: 1. in the lower segment nearest the site of i.m. injections the toxin could be detected almost immediately after injection; 2. in this same segment, the amount of toxin detected was proportional to the dosage used up to the 600 MLD level. Above this level the bioassay limits were exceeded (100~ T.E., death); 3. this maximal toxicity of the "stripped" nerve produced by dosage of 1000 MLD's or higher persisted in the left lower segment throughout the 24 h sampling period; 4. in contrast to the lower segment toxin was not detectable in the left middle and upper segments until 12 and 18 h after injection, respectively; 5. in these segments the toxin content was critically dosage dependent with the middle and upper nerve segments requiring a 2500 and 6000 MLD dosage level, respectively.

Discussion

Bilateral Distribution o/Toxin in Peripheral Nerves The detection of toxin in both sciatic nerves after intravenous and intramuscular toxin injections has been frequently reported using bioassay and labeled toxin methods (King). These observations have been used as strong evidence to support the I-lematogenous Dissemination Theory (Abel et al.). Abel et al. argued that the only reasonable mechanism for toxin to reach the uninjeeted extremity was via the vascular system. The bilateral distribution of toxin after injections, especially intramuscular, implied a vascular not neural mechanism of toxin distribution. If the bilateral toxin distribution is due to bloodborne toxin, then the Neuronal Toxin Transport Theory would appear tenuous. The mechanisms of local tetanus would then have to be examined on grounds other than neural transport of toxin to the spinal cord. l~ studies indicate that tetanus toxin was present initially only in the sciatic nerve from the injected extremity, primarily the lower nerve segment, nearest the site of toxin injection (King). However, allowing sufficient time (45 min--i h) for toxin distribution, the presence of toxin was detected in the undissected sciatic nerve from the opposite, uninjected extremity. We, as well as Kryzhanovskyii (1967a, b) have investigated the crucial role of the bilateral distribution of tetanus toxin in peripheral nerves on the mechanisms of local tetanus. Kryzhanovskyii (1967a) found that by injecting small intramuscular toxin dosages into rats (approximately i0 rat MLDs) that no toxin could be detected in undissected nerve homogenages from the uninjeeted extremity. I-Iowever, the intramuscularly injected toxin was detected in the 17 Act,a neuropath. (Berl.)~Bd.24

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homogenized nerve from the injected extremity. Kryzhanovskyii (1967a) was further able to detect a unilateral toxin distribution along the entire undissected sciatic nerve from the injected extremity in several species (rat, monkey, donkey, cat, rabbit). His experimental approach utilized the dilution effect that occurs when small amounts of i.m. injected toxin reach the much larger vascular pool. Since the intraneural blood vessels have a small volume, the effects of the vascular toxin would be proportionally decreased in the uninjected extremity. In our previous studies (King), the toxin dosage necessary to produce this distinct dissociation of toxin content between the two undissected sciatic nerves were approximately half of the dosage used by Kryzhanovskyii. This difference in dosage between these two studies might be due to differences in determining the concentration of tetanus toxin by bioassay of whole nerve homogenates instead of dissected nerve components and the use of the Percent Tetanus Effect method of toxicity determinations.

Epineurium and Bilateral Toxin Distribution In spite of its accessibility, the epineurium has not been extensively investigated recently in relation to the toxin distribution within the peripheral nerves. Most investigators have tacitly assumed that the epineurial lymphatic and blood vessels contain toxin after either intravenous or intramuscular toxin injections. The toxin distribution within the epineurinm with its lymphatics and blood vessels could explain the bilateral toxin distribution that occurs in the undissected nerves during the development of locM tetanus. In our experiments, the epineurinm was noted to be a significant site of toxicity after both intravenous and intramuscular injections (Groups IIc, d). Although a bilateral epineurial toxin distribution was noted, there were important differences. The epineurial toxin content of the lower nerve segment was always very different between extremities. For instance, when the toxin content of the injected extremity declined from its peak injection values, the toxin content of the uninjected extremity was slowly rising to a fairly stable level. Also, the undissected nerve's toxicity from the left lower segment was different from the epineurinm at the lower toxin dosages. This difference appeared to be due to a significant amount of "stripped" nerve toxin content. In the middle and upper segment, and all three segments from the uninjected extremity, the toxin content of the epinenrinm at 1 h was very low, but steadily increased until 12 h when it also plateaued. This slow absorption of toxin after i.m. injections has also been noted by Habermann using 125I-labeled toxin. Except for the left lower segment, the toxin content of the epineurinm from the uninjected and injected extremities was almost always comparable to the toxin content of the equivalent undissected nerve. The epineurinm from either extremity was not usually comparable to the "stripped" nerve from the same segment (Fig. 1). However, it was possible to saturate the left lower nerve segment such that the undissected nerve, epineurinm and "stripped" nerve were all uniformly lethal when toxin dosages were 1000 MLDs or higher. These data indicate that a bilateral toxin distribution noted in the undissected nerves could be produced by epineurial toxin. During the experimental period (1 h to 24 h), the distribution mechanisms that produced this difference

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in epineurial toxicity appeared to be acting at three different times. These changing distribution patterns were noted: 1. immediately after injection; 2. from 1 to 12 h after injection, and 3. from 12 to 24 h after injection. An exclusively vascular distribution mechanism could not account for these changing distribution patterns satisfactorily. The conclusions most compatible with our data were that the changing epineurial distribution patterns represented: 1. the intramuscular toxin injection and the injection forces, 2. lymphatic uptake and distribution of the injected toxin, and 3. intraneural blood vessels and tissue space toxin content. In the injected extremity, the initial high toxicity of the epineurium from the sciatic nerve segment appears to represent toxin spread via injection forces. Then, the continued high toxicity (1 to 12h) of the epineurium appeared to represent toxin leaving the injected muscles to reach the vascular system via the epineurial lymphatics. The subsequent similarity (12 to 24 h) of the epineurial toxin content in both extremities would then represent toxin primarily due to a blood vascular mechanism. Groups I I and I I b showed that even nerve transection did not prevent the epineurium of the remaining segments from both extremities fl'om acquiring toxin. In the uninjected extremity, the epineurium did not become toxic until after the bIood concentration had greatly increased. The subsequent epineurial toxicity appeared to reflect the dynamic equilibrium produced by intravascular, intralymphatic, and extracellular mechanisms. Thus, after intramuscular toxin injections, the epineurial toxin content of the uninjected extremity primarily represented lymphatic and blood distribution mechanisms. ]5~ear the injection site, the intravascular toxin did not appear to be g major determinant of the epineurial toxin content until the locally operating injection forces and lymphatic uptake mechanisms had declined. "Stripped Nerve". This bilateral epineurial toxin distribution (above) supported and clarified the often-reported toxin detection within both peripheral nerves after intramuscular toxin injections. However, when the epineurium was removed from the sciatic nerve segments, a different distribution of the intramuscularly injected toxin was observed. In the "stripped" nerve preparations, toxin was detected in the injected extremity, but not in the uninjected extremity (Fig. 1). To our knowledge, this is the first report of such unilateral distribution of toxin within different components of peripheral nerves after i.m. injections. I f the unilateral toxin distribution were to be considered valid, then several alternatives to neural transport had to be evaluated. Injury to the injected muscle that could cause bloodborne toxin to diffuse into the "stripped" sciatic nerve in the injected extremity was considered as a possible cause of this distribution. Unless this injm'y produces segmental or centripetal damage that occurs at differing rates, the distribution cannot be explained by bloodborne toxin diffusing into the "stripped" nerves. Likewise, the possibility that injection forces with resultant edema would cause the toxin distribution were considered. The uniform injection volumes, the negligible differences in oncotic forces due to toxin dosage differences, and the anatomy of the sciatic nerve passage through the pelvis to reach the iutervertebral spaces argue against the artifactual localization of toxin along the "stripped" nerve at different time intervals. The data in support of a neural transport of toxin along the "stripped" nerve from the injected extrem17"

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i t y was t h e n e v a l u a t e d . I n the i n j e c t e d e x t r e m i t y , t o x i n was d e t e c t e d in increasing a m o u n t s in each of t h e t h r e e " s t r i p p e d " nerve segments as t h e t o x i n dosage increased. As t h e s a m p l i n g i n t e r v a l increased, t o x i n was n o t e d in progressively higher (centripetal) " s t r i p p e d " nerve segments. These effects of increased t o x i n dosage a n d s a m p l i n g t i m e on t h e a c c u m u l a t i o n of t o x i n in " s t r i p p e d " nerve segments could be e l i m i n a t e d b y nerve t r a n s e c t i o n (Groups I I a, b). This d i s t r i b u t i o n p a t t e r n t h u s h a d t h e characteristics of a c e n t r i p e t a l t o x i n g r a d i e n t along t h e " s t r i p p e d " nerve in t h e i n j e c t e d e x t r e m i t y . T h a t this " t o x i n g r a d i e n t " occurred prior to t h e d e v e l o p m e n t of local t e t a n u s in t h e same e x t r e m i t y was especially n o t e w o r t h y (Fig. 1). H a b e r m a n n n o t e d t h a t 1~5I labelled t o x i n a) c o n c e n t r a t e d in t h e v e n t r a l roots, b) could be d e t e c t e d in this area before t h e t i m e s y m p t o m s of local t e t a n u s a p p e a r e d e) could n o t be d e t e c t e d a n d local t e t a n u s was p r e v e n t e d if the sciatic nerve was sectioned. The p r o p o s a l t h a t t o x i n could be t r a n s p o r t e d in t h e tissue spaces or axons to t h e spinal cord has been a m p l y s u p p o r t e d b y b o t h direct (above) a n d i n d i r e c t e x p e r i m e n t s using nerve ligation, nerve sclerosis, nerve splitting, a n d a n t i t o x i n blockage ( L a m a n n a a n d Cart; L a u r e n c e a n d W e b s t e r ; K r y z h a n o v s k y i i , 1967b; Fedinec, 1959, King).

Acknowledgements. This investigation was supported by USPHS grant AI-08610 from the National Institute of Allergy and Infectious Diseases, and NIH Training Grant No. G ~ 00202-10 from the National Institute of General Medical Sciences. Purified tetanus toxin and antitoxin was kindly provided and prepared by Mr. W. C. Latham, Institute of Laboratories, Commonwealth of Massachusetts Department of Public ttealth, Boston, Massachusetts. References Abel, J . J . , Evans, E.A., Jr., Hampil, B., Lee, F. C. : Researches on tetanus. II. The toxin of the bacillus tetani is not transported to the central nervous system by any component of the peripheral nerve trunks. Bull. John ttopk. Hosp. 56, 84--114 (1935). DahlstrSm, A.: Axoplasmie transport (with particular respect to adrenergie neurons). Phil. Trans. B 261, 325--358 (1971). Fedinec, A. A.: Absorption and distribution of tetanus toxin in experimental animals. In: Principles on tetanus, pp. 169--176. L. Eckmann, ed. Bern: H. Huber 1967. Fedinec, A.A., Matzke, H . A . : The relationship of toxin and antitoxin injection site to tetanus development in the rat. J. exp. Med. 110, 1023--1040 (1959). ~Iabermann, E.: Pharmakokinetisehe Besonderheiten des Tetanustexins und ihre Beziehungen zur P~thogenese des Iokalen bzw. generalisierten Tetanus. Naunyn-Schmiedebergs Arch. Pharmak. 267, 1--19 (1970). ttabermann, E.: Distribution of 125I-tet~nus toxin and 12~I-toxoid in rats with local tetanus, as influenced by antitoxin. Naunya-Sehmiedeberg's Arch. Pharmacol. 272, 75--88 (1972). Imbriano, A. E.: Determinaeion experimental de la dosis mortal media D.~. 50 de la toxin tets Su importancia y sus variaciones. Sen. m~d. (B. Aires) 57, 715--729 (1950). Karlsson, J. D., SjSstrand, J.: Transport of labelled proteins in the optic nerve and tract of the rabbit. Brain Res. 11, 431--439 (1968). King, L. E., Jr.: Distribution of tetanus toxin in the rat peripheral nerve components during the development of local tetanus. Ph.D., Thesis, University of Tennessee, Memphis, September 1970. Dissertation, Abst. Int. 82 (2), 1971. Kryzhanovskyii, G.N.: The neural pathway of toxin. In: Principles on tetanus, pp. 155 to 168. L. Eckmanu, ed. Bern: H. Huber 1967a. Kryzhanovskyii, G.N.: Detection of toxin in the nerves. In: Tetanus, pp. 104--125, Moscow: Meditsina 1967b.

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Lamanna, C., Carr, C. J." The botulinal, tetanus, and enterostaphlococcal toxins: A review. Clin. Pharmacol. Ther. 8, 286--332 (1967). Lasek, R.: Axoplasmie transport in eat dorsal root ganglion cells: As studied with aH-L-leucine. Brain Res. 7, 360--377 (1968). Latham, W.C., Jennesse, C.P., Timperi, R. J . K . , Michelsen, C. B.H., Zipilivan, E. M., Edsall, G., Ley, H. L., Jr.: Purification and characterization of tetanus toxoid and toxin. I. Fractionation of tetanus toxoid by gel filtration. J. Immunol. 95, 487--493 (1965). Laurence, D. R., Webster, R. A. : Pathologic physiology, pharmacology, and therapeutics of tetanus. Clin. Pharmacol. Ther. 4, 36--72 (1963). Marie, A , Morax, V.: Recherches sur l'absorption de la toxine t6tanique. Ann. Inst. Pasteur 16, 818--832 (1902). Ochs, S., Sabri, M. L, Johnson, J.: :Fast transport of materials in mammalian nerve fibers. Science 163, 686--687 (i969). Shantha, T. 1~., Bourne, G. H. : The perineural epithelimn--a new concept. In: The structure and function of nervous tissue, Vol. I, pp. 379--459. G. H. Bourne, ed. ~ew York: Academic Press 1968. Lloyd E. King, Jr., M.D. Ph.D. Veteran's Administration Hospital Electron Microscope Research Unit BECE-G-33, 1030 Jefferson Memphis, Tennessee, 38104 U.S.A.