TOXICOLOGY AND APPLIED PHARMACOLOGY 79,348-352 (1985)

Methylprednisolone Treatment of an Organophosphorus- induced Delayed Neuropathy’

Methylprednisolone Treatment of an Organophosphorus-Induced Delayed Neuropathy. BAKER, T., AND STANEC, A. (1985). Toxicol. Appl. Pharrnacol. 79, 348-352. Diisopropylfluo- rophosphate (DFP) injected into the femoral artery of cats causes a localized organophosphorus- induced delayed neuropathy (OPIDN). Gait disturbances develop in the treated leg 14 days after DFP exposure and reaches a maximum at 2 I to 28 days after DFP. In viva high-frequency conditioning of soleus motor nerve endings evokes stimulus-bound repetitive neural discharges (SBR) and an obligatory potentiation of the muscle contractile response (PTP). In this OPIDN model, SBR and PTP are maximally suppressed at 21 to 28 days after DFP. A high-dose regimen of methylprednisolone started 30 to 40 min after DFP exposure and lasting for 20 days prevented the development of OPIDN. In the methylprednisolone-DFP treated cats, SBR and PTP functions were not suppressed and not different from those in untreated normal cats. 0 1985 Academic Press. Inc.

The injection of the organophosphorus agent, diisopropylfluorophosphate (DFP), in the femoral artery of cats causes a localized organophosphorus-induced delayed neurop- athy (OPIDN) in the injected legs (Lowndes et al., 1974, 1975). At 21 to 28 days after DFP, maximal gait disturbances are observed.

In normal, healthy cats following stimu- lation of the soleus motor nerve at 400 Hz for 10 set, each single stimulation of the nerve evokes a repetitive discharge from its endings (Standaert, 1963, 1964). The dis- charge is known as stimulus-bound repetition (SBR) and causes an obligatory potentiation of the indirectly evoked muscle contractile response, known as post-tetanic potentiation (PTP). At the time of maximal gait distur- bances in cats with the localized OPIDN, a maximal loss of the capacity of cat soleus nerve endings to generate SBR and to evoke the PTP occurs (Lowndes et al., 1974, 1975; Baker et al., 1980). This loss correlates well with structural changes in the endings (Glazer et al., 1978; Drakontides et al., 1982).

A regimen utilizing the glucocorticoids, triamcinolone and methylprednisolone, pre- vents the development of this OPIDN (Baker

’ Preliminary data were presented at the April, 1984 meeting of the Federation of American Societies for Experimental Biology.

et al., 1982). Treatment with the glucocorti- coids starts immediately after DFP exposure. Treatment following accidental exposure to DFP, however, usually is not able to begin immediately after exposure, and these two glucorticoids may not always be available. Thus, the current study was conducted to determine if a glucocorticoid regimen started 30 to 40 min after DFP and employing only one of the glucocorticoids would also be effective in preventing the OPIDN.

METHODS

This method has been well described previously (Stan- daert, 1963; Riker ef al., 1975). Briefly, a laminectomy and an in vivo soleus nerve-muscle preparation were performed in cats anesthetized with cr-chloralose, 90 mg/ kg, iv. SBR and PTP were, respectively, recorded from ventral root filaments containing one to three soleus axons and isometric muscle contractile responses. The soleus nerve was continuously stimulated with supra- maximal rectangular pulses of 0.1 msec duration at 0.4 Hz except when interrupted for IO-set trains at 400 Hz. After the 400 Hz/l0 set conditioning, the incidence of SBR and the area of the PTP responses were determined.

DFP, 2 mg/kg, was aseptically injected in a volume of 0.1 ml/kg in the left femoral artery of eight cats anesthetized with sodium pentobarbital, 25 mg/kg iv. Six of these cats received the following regimen of methylprednisolone: a 90 m&kg dose injected into the femoral vein of the contralateral leg 30 to 40 min after DFP administration, and an 8 mg/kg dose injected into

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the suprascapular muscles starting 24 hr later and repeated once every 3 days during the next 19 days, alternating the sites of injection between the left and right body sides. This resulted in each cat receiving eight methyl- prednisolone injections; for the iv injection, a water soluble form (methylprednisolone sodium succinate, Solu- Medrol) was used, while for the seven im injections, a muscle depot form (methylprednisolone acetate, Depo- Medrol) was used.

SBR and mP were evaluated in four of the DFP- methylprednisolone-treated cats 2 1 to 23 days after DFP exposure. In the 2 remaining methylprednisolone treated cats, SBR and PTP were evaluated at 70 days after DFP exposure. The 2 cats which received DFP and no meth- ylprednisolone served as DFP controls; SBR and PTP were evaluated at 21 days in the first cat and at 23 days in the second cat. The effect of DFP on these two cats established that the DFP was active and its effects on SBR and PTP at this time were the same as previously reported (Lowndes ez al., 1974; Baker ef a[., 1980, 1982; Drakontides and Baker, 1983). Since SBR and PTP values developed by normal healthy cats have been well established (Standaert, 1963; Baker et al., 1977, 1982), the untreated normal control values used in this study were taken from previous studies (Baker et al., 1980, 1982; Drakontides and Baker, 1983).

The Chi-square and Mann-Whitney tests were, re- spectively, used to analyze the SBR and PTP ratio data. Results were considered significant at p 6 0.05.

RESULTS

At 21 days after drug administration, the two cats which had received only DFP had a pronounced high-step gait in the treated leg with some compromise in jumping and landing abilities. These are the typical clinical manifestations of the OPIDN in this model (Lowndes et al., 1974, 1975; Baker and Lowndes, 1980; Baker et al., 1980, 1982). Figure 1B shows the loss in the SBR and PTP responses in one of the cats treated only with DFP. The degree of loss is typical of that seen in this OPIDN model. The inci- dence of SBR in these two cats was 9.8%: 4 of 40 axons exhibited SBR in the first cat, evaluated at 21 days after DFP, and 4 of 42 axons in the second cat, evaluated at 23 days after DFP. The PTP loss in the delayed neuropathy occurred not only in the peak tension but also in its duration. The ratio of PTP in these two DFP treated animals to

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FIG. 1. Effects of methylprednisolone treatment on typical stimulus-bound repetition (SBR) generated by cat soleus motor nerve endings and the obligatory potentiated muscle responses. Neural action potentials are shown in the left column and muscle contractile responses in the right column. At 1, the single action potential evoked by a single stimulus of the sciatic nerve and the muscle contractile response, At 2, the single action potential and the SBR train, if present, evoked by a single stimulus after 400 Hz/ 10 set conditioning. (A) Untreated, normal control; (B) Treated with diisopropylfluorophosphate (DFP), 2 mg/kg ia, 2 1 days earlier; (C) DFP administered 2 1 days earlier followed by methylprednisolone regimen (see Methods).

untreated normal controls was 0.388 (Table 1). Both the SBR incidence and the PTP loss agree with reported values for this OPIDN model (Lowndes et al., 1974; Baker et al., 1980, 1982; Drakontides and Baker, 1983). The typical SBR and PTP which occur in untreated normal cats are illustrated in Fig. 1A. The differences in the SBR incidences and the PTP ratios between DFP only treated and untreated normal cats are highly signifi- cant (Table 1).

None of the six DFP-treated cats which received methylprednisolone developed any neurotoxic signs of the OPIDN. Up to the time of SBR and PTP evaluation (21, 23, and 70 days), all animals jumped and landed in a well-coordinated manner without any

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TABLE 1

THE INCIDENCE OF STIMULUS-BOUND REPETITION (SBR) INDIRECTLY EVOKED BY SINGLE STIMULI AFTER 400

Hz/l0 set CONDITIONING AND THE RATIO OF THE OBLIGATORY POST-TETANIC POTENTIATION (PTP)

No. of No. of axons

axons demonstrating Cat no. tested SBR % PTP ratio”

(A) Diisopropylfluorophosphate (DPP) 2 mg/kg intraarterial (ia) 21 to 23 days earlier

I 40 4 10.0 0.390 2 42 4 9.5 0.386 Total 82 8 9.8' 0.388'

(B) Untreated normal controls: data of Baker et al. (1980, 1982); Drakontides et al. (1983); N = 16

711 586 82.4 1.00 AZ 0.029 (SE)

(C. 1) DPP, 2 mg/kg ia, 2 1 to 23 days earlier + methylprednisolone regimen’

3 50 41 82.0 1.05 4 50 40 80.0 1.10 5 49 42 85.7 1.08 6 51 43 84.3 1.01 Total 200 166 83.0d 1.06*

(C.2) DPP, 2 mg/kg ia, 70 days earlier + methyiprednisolone regimen’ during first 20 days

I 50 41 82.0 1.04 8 50 40 80.0 1.02 Total 100 81 81.0d 1.03d

4 Areas of PTP in treated cats/areas of PTP in untreated normal controls. ’ Significantly different from B (untreated normal controls), p < 0.001. c Methylprednisolone, 90 mg/kg iv, into the contralateral leg 30 to 40 min after DF’P and then 8 mg/kg im once

every 3 days from the 2nd to the 20th day after DFP. *Significantly different from A (treated only with DPP), p < 0.00 1.

favoring of the DFP-treated leg. In their motor movements and coordination, these cats could not be distinguished from un- treated normal cats. Two of these cats were evaluated 21 days after DFP administration and another two cats at 23 days after DIV. This period is the time at which the greatest losses of SBR and PTP occur in cats that receive only DPP. The last two DPP-meth- ylprednisolone-treated cats were evaluated for SBR and PTP after being observed for 70 days after DPP administration for clinical neurotoxic signs. Since none had developed by this time and the cats appeared normal in all their activities, SBR and PTP in these two cats were evaluated at this time.

In the six animals which received both DFP and the methylprednisolone regimen,

the SBR incidence and PTP ratio were the same as those in untreated normal controls (Table 1). In the 21 to 23&y animals, the SBR incidence and PTP ratio were 83.0% and 1.06, respectively; in the 7O-day cats, they were 81 .O% and 1.03. The combined values were 82.3% and 1.05. These are typical values for untreated normal controls and are signifkantly different from those of cats which received DFP alone.

DISCUSSION

The two cats which received the single intraarterial dose of DF’P developed the clin- ical signs of the OPIDN. The capacity of the cat soleus motor nerve endings to generate

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SBR and the obligatory PTP was maximally suppressed at 21 to 23 days after DFP ex- posure. Morphological damage to terminal structures in cat soleus motor nerves occurs at this time and correlates well with the loss of the SBR function (Glazer et al., 1978; Drakontides et al., 1982).

To determine that the OPIDN did not develop at a time later than that observed in cats treated only with DFP, two cats which received the intensive methylprednisolone 30 to 40 min after DFP exposure were observed for 70 days. None of these animals developed any clinical signs of the OPIDN. Cats which have been administered only DFP are near full recovery by 56 days after exposure: all signs of the neuropathy have disappeared and both SBR and PTP are near normal values (Lowndes et al., 1974, 1975). Since the cats which received both DFP and meth- ylprednisolone did not develop any neuro- toxic signs of the OPIDN by 70 days, it is highly unlikely that the OPIDN will develop after this time.

The incidences of SBR and the PTP ratios in the methylprednisolone treated DFP cats at 21 to 23 and 70 days after DFP are the same as in untreated normal cats. A similar high-dose regimen of methylprednisolone and triamcinolone prevents any morphological damage in this OPIDN model at 21 to 23 days after DFP (Baker et al., 1982; Drakon- tides et al., 1982). The functional and clinical evaluations clearly establish that the meth- ylprednisolone regimen prevents OPIDN, probably by preventing any structural changes in the endings. Evidently, glucocorticoids have the ability to prevent other neural losses. Pretreatment with glucocorticoids prevents OPIDN in hens (Glees, 196 I), motor impair- ment due to caudate nucleus damage (Beyer- Meat-s and Barnett, 1980), and loss of neu- romuscular transmission at 48 hr following nerve section (Hall et al., 1983).

The mechanisms by which glucocorticoids prevent OPIDN in this model are not eluci- dated by the design of the current experi- ments. Certain possible mechanisms, how-

ever, are suggested: (1) Spinal cord neurons have glucocorticoid receptors (Clark et al., 198 1). The glucocorticoid regimen could re- sult in an increased synthesis of substances essential for the maintenance of axonal and terminal integrity. (2) The glucocorticoid reg- imen could maintain membrane stability by reducing the activities of various lysosomal acid hydrolases (Koenig et al., 1980). (3) Neurotoxic esterase (NTE), a membrane bound enzyme, has been identified in periph- eral nerves (Lotti and Johnson, 1980). The glucocorticoid regimen could result in the synthesis of new NTE which would replace the organophosphorylated NTE, thereby pre- venting the sequence of events that result in the OPIDN. (4) The glucocorticoids, being highly lipid soluble, enter the membrane and could interfere directly with the sequelae of the phosphorylation of NTE, thereby pre- venting the development of OPIDN.

Currently, specific knowledge of the mo- lecular events in neurons caused by gluco- corticoids is insufficient to allow the identi- fication of the mechanisms by which these steroids prevent the development of OPIDN. This should not, however, prevent their pos- sible application in the treatment of OPIDN and similar neuropathies.

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BAKER, T., LOWNDES, H. E., JOHNSON, M. K., AND

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THOMASBAKER ANNA STANEC

Department of Anesthesiology St. Joseph’s Hospital and Medical Center 703 Main Street Paterson, New Jersey 07503

Received October 4. I984