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DFP mononeuropathy: Evidence for a peripheral site of initiation
248
Brain Research, 184 (1980) 248-251 © Elsevier/North-Holland Biomedical Press
DFP mononeuropathy: evidence for a peripheral site of initiation
RICHARD D. HOWLAND, HERBERT E. LOWNDES, THOMAS BAKER* and RUDY J. RICHARDSON** Department of Pharmacology, New Jersey Medical School, 100 Bergen Street, Newark, N. J., 07103 (u.s.A.)
(Accepted October 25th, 1979) Key words: DFP mononeuropathy - - peripheral neurotoxicity - - distal axonopathy
The foci at which neurotoxic organophosphorous compounds induce axonopathies have been thought to be the neural perikarya, with subsequent retrograde axonal degeneration resulting from interference with metabolic function in the soma 5. However, recent studies raise the possibility that the organophosphorous 3,4 and other neurotoxic agentsT,9,10 impact directly on the axon and the perikarya are not involved in the etiology of the axonopathy. A major difficulty in determining sites of initiation of axonal degeneration results from systemic administration of the toxic agent. A technique to circumvent this difficulty is to produce a localized, mononeuropathy by restricting the distribution of the neurotoxic agent. Diisopropylfluorophosphate (DFP) injection into one femoral artery of cats results in a mononeuropathy in the injected hindlimb, confirmed by physiologicaP,2, 8 and morphological2, 6 criteria. However, these studies do not definitely rule out perikaryal involvement in the neuropathy since the distribution of D F P following intraarterial injection is unknown. The present studies confirm that D F P is largely restricted to the injected leg, with relatively small amounts reaching the contralateral limb or the spinal cord. Ten adult mongrel cats were anesthetized with sodium pentobarbital (25 mg/kg i.v.), the left femoral artery exposed, and D F P (K and K Labs) (2 mg/kg) injected by means of a 30-gauge needle just below the level of the superficial epigastric artery. [3H]DFP (spec. act. 3.4 Ci/mmol, Amersham and Searle) was added to the injection solution to achieve approximately 20 × l0 Gcpm/kg body weight. Concentrations of solutions were adjusted so that the desired dosage was injected in a volume of 0.1 ml/kg body weight. An equal volume of saline was injected into the contralateral femoral artery. All solutions were freshly prepared just prior to injection. * Present address: Dept. of Pharmacology, Cornell University Medical College, New York, N.Y., U.S.A. ** Present address : Dept. of Environmental and Industrial Health, Toxicology Research Laboratory, University of Michigan School of Public Health, Ann Arbor, Mich. 48109, U.S.A.
249
350-
i
300-
2
250-
%
=~200-
m I
[~
DFP-Treated Side
~
Contratateral Side
TI
150-
10050(2
2 Distal~.
4 Sciatic
6
8 10 12 Nerve Segments (cm)
14
16 • • Dorsal Roots
Ventral Roots
Spinal Cord
Fig. 1. Distribution of D F P in cat spinal cord, spinal roots and peripheral nerve 1 h after unilateral injection into a femoral artery. D F P concentrations (open bars, injected side; hatched bars, contra]ateral side) calculated from cpm o f tritiated D F P in hemisections of spinal cord (L7-SI), dorsal and ventral roots, and in 1 cm segments of nerve starting with the tibial nerve at the ankle and c o n t i n u i n g u p the sciatic nerve. T h e fifth c m s e g m e n t is the point at which the triceps surae nerve
bifurcates from the sciatic nerve. Values are the mean ± S.E.M. from 5-6 animals and are cpm/g wet weight of tissue.
Both sciatic nerves were removed under pentobarbital anesthesia 1 or 24 h after D F P administration, along with the tibial nerve to the ankle, the triceps surae nerves, dorsal and ventral roots L7 and S 1 and the corresponding segments of the spinal cord. Samples of lateral gastrocnemius and soleus muscles and blood samples were also taken bilaterally. The nerves were cut into 1 cm lengths and the spinal cord divided longitudinally into right and left halves. All tissues were weighed and solubilized in NCS (Nuclear Chicago). Ten ml of toluene-based scintillation cocktail (LSC complete, Yorktown) together with 50 ~1 of glacial acetic acid to quench chemiluminescence were added and the samples counted on an Intertechnique Liquid scintillation counter (Model PG-4000). All values are corrected for tissue blanks obtained from 3 noninjected cats. One hour after injection the radioactivity in the sciatic nerve of the injected limb showed a proximal-distal gradient, the higher concentrations being present in the more distal segments of the nerve (Fig. l). The highest level of radioactivity (35,300 45,500 cpm/g; mean ± S.E.M.) was observed in the segment 5 cm proximal to the ankle which corresponds to a D F P concentration of 2.49 4- 0.33 /zg/g (mean 4S.E.M.). In contrast, radioactivity in the sciatic nerve of the contralateral side was
250 uniformly distributed along the entire nerve trunk (Fig. 1). The mean cpm/g for all nerve segments from the non-injected limb was 3,720 ~ 160. No difference in the levels of radioactivity were observed between the dorsal roots, ventral roots or spinal cord obtained from either side (Fig. 1). The radioactivity in the injected and contralateral sides of the spinal cord corresponded to 0.37 ± 0.03 and 0.38 -+- 0.04/~g DFP/g, respectively (mean -+- S.E.M.). Significant levels of radioactivity were found in the medial gastrocnemius and soleus muscles of the DFPtreated limb (Table I), whereas only low levels were found in the muscles of the contralateral limb and were similar to those in the sciatic nerve. The triceps surae nerve from the DFP-treated limb contained 33,700 -~ 7,200 cpm/g, a level of radioactivity comparable to the peak levels in the sciatic nerve (Table I). Serum levels of radioactivity were not significantly different from each other. The distribution of radioactivity along the sciatic nerve of the injected limb 24 h post-injection was qualitatively similar to the earlier time period. A proximal-distal gradient was observed with peak levels being present in the segments 5 and 6 from the ankle (7170 and 7010 cpm/g, respectively). As in the 1 h data, no differences were observed in the amount of label of the injected vs non-injected side for spinal cord, dorsal roots and ventral roots. Levels of radioactivity, however, were approximately 25-35 ~ of the 1 h levels. The radioactivity for the triceps surae nerve, medial gastrocnemius and soleus muscles and serum 24 h after injection are given in Table I and show that except for the reduction in level, the relative distribution remains the same. These data confirm that D F P is not significantly redistributed. The concentrations of D F P in the spinal cord are not significantly greater than in the corresponding spinal roots on the injected side (Fig. 1). Equal amounts of label appear in both sides of the cord, exposing the anterior horn cells innervating muscles in both the injected and contralateral limbs to equal amounts of the toxic agent. If the perikarya are involved in the genesis of this mononeuropathy, it would then become manifest in both limbs. No functional changes are observed in the contralateral limb at
TABLE I Radioactivity in tissues 1 or 24 h after injection o f [3H]DFP into one femoral artery
Values are mean ± S.E.M. Numbers in parentheses are the number of animals investigated. Tissue
cpm/g × 10 -2 1h Injected
Triceps surae nerve Medial gastrocnemius muscle Soleus muscle Serum (cpm/ml)
24h Contralateral
Injected
Contralateral
10.4 - - 11.0"
337 ± 72 (6)
34 ± 7 (3)
30.9 -- 48.0*
196 ~ 59 (6) 167 ± 44 (6) 340 ± 32 (5)
37 ± 5 (6) 31 ± 6 (5) 272 ~ 46 (5)
27 ± 3 (3) 56 ± 22 (3) 155 ± 71 (3)
* Range of 2 determinations.
11.2 £- 1.3 (3) 13.9 ± 1.1 (3) 135 ± 41 (3)
251 a n y time after D F P a d m i n i s t r a t i o n1,z,s. Further, if D F P causes this distal a x o n o p a t h y by i m p a i r i n g perikaryal function, equivalent pathological changes should a p p e a r in m o t o r nerve terminals of the same m o t o r unit 6. It c a n n o t be concluded o n the basis of the present d a t a or earlier studies whether D F P exerts its neurotoxic effects directly on m o t o r nerve terminals, i n t r a m u s c u l a r branches or more proximal p o r t i o n s of the nerve. F u r t h e r m o r e , damage to other tissues, such as muscle or S c h w a n n cells in the etiology of D F P n e u r o p a t h y c a n n o t be determined by these data. The u n e q u a l pathology observed in nerve terminals o f the same m o t o r u n i t following D F P injection6, however, is consistent with the damage at b r a n c h i n g points o f m o t o r axons observed in vitro with 2,5-hexanedione11,12. The present data s u p p o r t the c o n t e n t i o n of B o u l d i n a n d Cavanagh3, 4 that D F P c a n cause a 'chemical t r a n s e c t i o n ' by a direct toxic action o n axons. This work was supported by U S P H S G r a n t s NS-01447 a n d NS-11948.
1 Baker T. and Lowndes H. E., Muscle spindle function in delayed organophosphorus neuropathy, Brain Research, in press. 2 Baker T., Glazer E. and Lowndes H. E., Subacute neuropathic effect of diisopropylfluorophosphate at the cat soleus neuromuscular junction, NeuropathoL appl. NeurobioL, 3 (1977) 337-350. 3 Bouldin T. W. and Cavanagh J. B., Organophosphorous neuropathy. I. A teased fiber study of the spatio-temporal spread of axonal degeneration, Amer. J. PathoL, 94 (1979) 241-252. 4 Bouldin T. W. and Cavanagh J. B., Organopbosphorous neuropathy. II. A fine-structural study of the early stages of axonal degeneration, Amer. J. PathoL, 94 (1979) 253-271. 5 Cavanagh J. B., The significance of the 'dying-back' process in experimental and human neurological disease, Int. Rev. exp. PathoL, 3 (1964) 219-267. 6 Glazer E. J., Baker T. and Riker W. F., Jr., The neuropathy of DFP at cat soleus neuromuscular junction, J. Neurocytol., 7 (1978) 741-758. 7 Lowndes H. E. and Baker T., Toxic site of action in distal axonopathies. In P. S. Spencer and H. H. Schaumburg (Eds.), Clinical and Experimental Neurotoxicology, Williams and Wilkins, Baltimore, in press. 8 Lowndes H. E., Baker T. and Riker W. F., Jr., Motor nerve dysfunction in delayed DFP neuropathy, Europ. J. PharmacoL, 29 (1974) 66-73. 9 Schaumburg H. H., Wisniewski H. M. and Spencer P. S., Ultrastructural studies of the dyingback process. I. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication, J. Neuropath. exp. Neurol. 33 (1974) 260-284. 10 Spencer P. S. and Schaumburg H. H., Pathobiology of neurotoxic axonal degeneration. In S. Waxman (Ed), Physiology and Pathobiology of Axons, Raven press, New York, 1978, pp. 265-282. 11 Spencer P. S., Schaumburg, H. H. and Peterson E. R., Neurofilamentous axonal degeneration in vivo and in vitro produced by 2,5-hexanedione, Neurosci. Abstr., 1 (1975) 703. 12 Veronesi B., Peterson E., DiVincenzo G. and Spencer P. S., A tissue culture model of distal (dying-back) axonopathy: its use in determining primary neurotoxic hexacarbon compounds, J. Neuropath. Exp. Neurol., 37 (1978) 703.
Brain Research, 184 (1980) 248-251 © Elsevier/North-Holland Biomedical Press
DFP mononeuropathy: evidence for a peripheral site of initiation
RICHARD D. HOWLAND, HERBERT E. LOWNDES, THOMAS BAKER* and RUDY J. RICHARDSON** Department of Pharmacology, New Jersey Medical School, 100 Bergen Street, Newark, N. J., 07103 (u.s.A.)
(Accepted October 25th, 1979) Key words: DFP mononeuropathy - - peripheral neurotoxicity - - distal axonopathy
The foci at which neurotoxic organophosphorous compounds induce axonopathies have been thought to be the neural perikarya, with subsequent retrograde axonal degeneration resulting from interference with metabolic function in the soma 5. However, recent studies raise the possibility that the organophosphorous 3,4 and other neurotoxic agentsT,9,10 impact directly on the axon and the perikarya are not involved in the etiology of the axonopathy. A major difficulty in determining sites of initiation of axonal degeneration results from systemic administration of the toxic agent. A technique to circumvent this difficulty is to produce a localized, mononeuropathy by restricting the distribution of the neurotoxic agent. Diisopropylfluorophosphate (DFP) injection into one femoral artery of cats results in a mononeuropathy in the injected hindlimb, confirmed by physiologicaP,2, 8 and morphological2, 6 criteria. However, these studies do not definitely rule out perikaryal involvement in the neuropathy since the distribution of D F P following intraarterial injection is unknown. The present studies confirm that D F P is largely restricted to the injected leg, with relatively small amounts reaching the contralateral limb or the spinal cord. Ten adult mongrel cats were anesthetized with sodium pentobarbital (25 mg/kg i.v.), the left femoral artery exposed, and D F P (K and K Labs) (2 mg/kg) injected by means of a 30-gauge needle just below the level of the superficial epigastric artery. [3H]DFP (spec. act. 3.4 Ci/mmol, Amersham and Searle) was added to the injection solution to achieve approximately 20 × l0 Gcpm/kg body weight. Concentrations of solutions were adjusted so that the desired dosage was injected in a volume of 0.1 ml/kg body weight. An equal volume of saline was injected into the contralateral femoral artery. All solutions were freshly prepared just prior to injection. * Present address: Dept. of Pharmacology, Cornell University Medical College, New York, N.Y., U.S.A. ** Present address : Dept. of Environmental and Industrial Health, Toxicology Research Laboratory, University of Michigan School of Public Health, Ann Arbor, Mich. 48109, U.S.A.
249
350-
i
300-
2
250-
%
=~200-
m I
[~
DFP-Treated Side
~
Contratateral Side
TI
150-
10050(2
2 Distal~.
4 Sciatic
6
8 10 12 Nerve Segments (cm)
14
16 • • Dorsal Roots
Ventral Roots
Spinal Cord
Fig. 1. Distribution of D F P in cat spinal cord, spinal roots and peripheral nerve 1 h after unilateral injection into a femoral artery. D F P concentrations (open bars, injected side; hatched bars, contra]ateral side) calculated from cpm o f tritiated D F P in hemisections of spinal cord (L7-SI), dorsal and ventral roots, and in 1 cm segments of nerve starting with the tibial nerve at the ankle and c o n t i n u i n g u p the sciatic nerve. T h e fifth c m s e g m e n t is the point at which the triceps surae nerve
bifurcates from the sciatic nerve. Values are the mean ± S.E.M. from 5-6 animals and are cpm/g wet weight of tissue.
Both sciatic nerves were removed under pentobarbital anesthesia 1 or 24 h after D F P administration, along with the tibial nerve to the ankle, the triceps surae nerves, dorsal and ventral roots L7 and S 1 and the corresponding segments of the spinal cord. Samples of lateral gastrocnemius and soleus muscles and blood samples were also taken bilaterally. The nerves were cut into 1 cm lengths and the spinal cord divided longitudinally into right and left halves. All tissues were weighed and solubilized in NCS (Nuclear Chicago). Ten ml of toluene-based scintillation cocktail (LSC complete, Yorktown) together with 50 ~1 of glacial acetic acid to quench chemiluminescence were added and the samples counted on an Intertechnique Liquid scintillation counter (Model PG-4000). All values are corrected for tissue blanks obtained from 3 noninjected cats. One hour after injection the radioactivity in the sciatic nerve of the injected limb showed a proximal-distal gradient, the higher concentrations being present in the more distal segments of the nerve (Fig. l). The highest level of radioactivity (35,300 45,500 cpm/g; mean ± S.E.M.) was observed in the segment 5 cm proximal to the ankle which corresponds to a D F P concentration of 2.49 4- 0.33 /zg/g (mean 4S.E.M.). In contrast, radioactivity in the sciatic nerve of the contralateral side was
250 uniformly distributed along the entire nerve trunk (Fig. 1). The mean cpm/g for all nerve segments from the non-injected limb was 3,720 ~ 160. No difference in the levels of radioactivity were observed between the dorsal roots, ventral roots or spinal cord obtained from either side (Fig. 1). The radioactivity in the injected and contralateral sides of the spinal cord corresponded to 0.37 ± 0.03 and 0.38 -+- 0.04/~g DFP/g, respectively (mean -+- S.E.M.). Significant levels of radioactivity were found in the medial gastrocnemius and soleus muscles of the DFPtreated limb (Table I), whereas only low levels were found in the muscles of the contralateral limb and were similar to those in the sciatic nerve. The triceps surae nerve from the DFP-treated limb contained 33,700 -~ 7,200 cpm/g, a level of radioactivity comparable to the peak levels in the sciatic nerve (Table I). Serum levels of radioactivity were not significantly different from each other. The distribution of radioactivity along the sciatic nerve of the injected limb 24 h post-injection was qualitatively similar to the earlier time period. A proximal-distal gradient was observed with peak levels being present in the segments 5 and 6 from the ankle (7170 and 7010 cpm/g, respectively). As in the 1 h data, no differences were observed in the amount of label of the injected vs non-injected side for spinal cord, dorsal roots and ventral roots. Levels of radioactivity, however, were approximately 25-35 ~ of the 1 h levels. The radioactivity for the triceps surae nerve, medial gastrocnemius and soleus muscles and serum 24 h after injection are given in Table I and show that except for the reduction in level, the relative distribution remains the same. These data confirm that D F P is not significantly redistributed. The concentrations of D F P in the spinal cord are not significantly greater than in the corresponding spinal roots on the injected side (Fig. 1). Equal amounts of label appear in both sides of the cord, exposing the anterior horn cells innervating muscles in both the injected and contralateral limbs to equal amounts of the toxic agent. If the perikarya are involved in the genesis of this mononeuropathy, it would then become manifest in both limbs. No functional changes are observed in the contralateral limb at
TABLE I Radioactivity in tissues 1 or 24 h after injection o f [3H]DFP into one femoral artery
Values are mean ± S.E.M. Numbers in parentheses are the number of animals investigated. Tissue
cpm/g × 10 -2 1h Injected
Triceps surae nerve Medial gastrocnemius muscle Soleus muscle Serum (cpm/ml)
24h Contralateral
Injected
Contralateral
10.4 - - 11.0"
337 ± 72 (6)
34 ± 7 (3)
30.9 -- 48.0*
196 ~ 59 (6) 167 ± 44 (6) 340 ± 32 (5)
37 ± 5 (6) 31 ± 6 (5) 272 ~ 46 (5)
27 ± 3 (3) 56 ± 22 (3) 155 ± 71 (3)
* Range of 2 determinations.
11.2 £- 1.3 (3) 13.9 ± 1.1 (3) 135 ± 41 (3)
251 a n y time after D F P a d m i n i s t r a t i o n1,z,s. Further, if D F P causes this distal a x o n o p a t h y by i m p a i r i n g perikaryal function, equivalent pathological changes should a p p e a r in m o t o r nerve terminals of the same m o t o r unit 6. It c a n n o t be concluded o n the basis of the present d a t a or earlier studies whether D F P exerts its neurotoxic effects directly on m o t o r nerve terminals, i n t r a m u s c u l a r branches or more proximal p o r t i o n s of the nerve. F u r t h e r m o r e , damage to other tissues, such as muscle or S c h w a n n cells in the etiology of D F P n e u r o p a t h y c a n n o t be determined by these data. The u n e q u a l pathology observed in nerve terminals o f the same m o t o r u n i t following D F P injection6, however, is consistent with the damage at b r a n c h i n g points o f m o t o r axons observed in vitro with 2,5-hexanedione11,12. The present data s u p p o r t the c o n t e n t i o n of B o u l d i n a n d Cavanagh3, 4 that D F P c a n cause a 'chemical t r a n s e c t i o n ' by a direct toxic action o n axons. This work was supported by U S P H S G r a n t s NS-01447 a n d NS-11948.
1 Baker T. and Lowndes H. E., Muscle spindle function in delayed organophosphorus neuropathy, Brain Research, in press. 2 Baker T., Glazer E. and Lowndes H. E., Subacute neuropathic effect of diisopropylfluorophosphate at the cat soleus neuromuscular junction, NeuropathoL appl. NeurobioL, 3 (1977) 337-350. 3 Bouldin T. W. and Cavanagh J. B., Organophosphorous neuropathy. I. A teased fiber study of the spatio-temporal spread of axonal degeneration, Amer. J. PathoL, 94 (1979) 241-252. 4 Bouldin T. W. and Cavanagh J. B., Organopbosphorous neuropathy. II. A fine-structural study of the early stages of axonal degeneration, Amer. J. PathoL, 94 (1979) 253-271. 5 Cavanagh J. B., The significance of the 'dying-back' process in experimental and human neurological disease, Int. Rev. exp. PathoL, 3 (1964) 219-267. 6 Glazer E. J., Baker T. and Riker W. F., Jr., The neuropathy of DFP at cat soleus neuromuscular junction, J. Neurocytol., 7 (1978) 741-758. 7 Lowndes H. E. and Baker T., Toxic site of action in distal axonopathies. In P. S. Spencer and H. H. Schaumburg (Eds.), Clinical and Experimental Neurotoxicology, Williams and Wilkins, Baltimore, in press. 8 Lowndes H. E., Baker T. and Riker W. F., Jr., Motor nerve dysfunction in delayed DFP neuropathy, Europ. J. PharmacoL, 29 (1974) 66-73. 9 Schaumburg H. H., Wisniewski H. M. and Spencer P. S., Ultrastructural studies of the dyingback process. I. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication, J. Neuropath. exp. Neurol. 33 (1974) 260-284. 10 Spencer P. S. and Schaumburg H. H., Pathobiology of neurotoxic axonal degeneration. In S. Waxman (Ed), Physiology and Pathobiology of Axons, Raven press, New York, 1978, pp. 265-282. 11 Spencer P. S., Schaumburg, H. H. and Peterson E. R., Neurofilamentous axonal degeneration in vivo and in vitro produced by 2,5-hexanedione, Neurosci. Abstr., 1 (1975) 703. 12 Veronesi B., Peterson E., DiVincenzo G. and Spencer P. S., A tissue culture model of distal (dying-back) axonopathy: its use in determining primary neurotoxic hexacarbon compounds, J. Neuropath. Exp. Neurol., 37 (1978) 703.