Rapid transneuronal destruction following peripheral nerve transection in the medullary dorsal horn is enhanced by strychnine, picrotoxin and bicuculline

385

Pain, 30 (1987) 385-393 Elsevier

PAI 01086

Rapid transneuronal destruction following peripheral nerve transection in the medullary dorsal horn is enhanced by strychnine, picrotoxin and bicuculline

Tomosada Sugimoto, Motohide Takemura, Akira Sakai and Masashi Ishimaru * Second Department of Oral Anatomy and * Research Resource Center, Osaka University Faculty of Dentistry, l-8 Yamadaoka, Suita, Osaka 565 (Japan) (Received

30 October

1986, revised received

10 February

1987, accepted

11 February

1987)

The effects of systemic administration of strychnine (1 mg/kg), picrotoxin (0.5 mg/kg) Summary and bicuculline (2 mg/kg) on acute transsynaptic destruction of medullary dorsal horn neurons following transection of the inferior alveolar nerve were assessed in rats. Single intraperitoneal injections of the above drugs were given without, 1 min before or 1 min after the nerve transection. The effect of transection without drug administration was also examined. Eighteen hours after nerve transection without drug, approximately 7 dark neurons were found in a single toluidine blue stained 1 nm section of the rostra1 medullary dorsal horn ipsilateral to the nerve transection. Administration of the drugs 1 min before the nerve transection significantly increased the number of dark neurons in a single section to about 17 (strychnine), 46 (picrotoxin) and 20 (bicuculline). These dark neurons were found mainly in the dorsal half of medullary dorsal horn. Delivery of any of the drugs 1 min after the nerve transection did not increase the number of dark neurons. The data thus indicate that the transneuronal effect of transection of the nerve was enhanced by antagonism of glycinergic and GABAergic inhibition of dorsal horn neurons. In view of the short latency and duration of transsynaptic destructive activity, a massive inJury discharge of primary afferent neurons and the subsequent release of excitatory neurotransmitters appear to be the direct cause of convulsant-enhanced rapid transsynaptic destruction which follows the peripheral nerve transection.

Key words: Peripheral

nerve; Dorsal

horn;

Strychnine;

Picrotoxin;

Correspondence to: Dr. T. Sugimoto, Osaka University Oral Anatomy, l-8 Yamadaoka, Suita, Osaka 565, Japan.

0304-3959/87/$03.50

0 1987 Elsevier Science Publishers

Faculty

Bicuculline

of Dentistry,

B.V. (Biomedical

Second

Division)

Department

of

386

Introduction Chronic peripheral nerve injuries in adult mammals have been known to cause transneuronal destruction of dendrites in the dorsal horn neurons [9,24,25]. This transneuronal destruction, however, did not ultimately lead to destruction of cell bodies unless it was enhanced by systemic administration of strychnine [25]. Since strychnine is a potent antagonist of glycine which is thought to mediate the postsynaptic inhibition of dorsal horn neurons, the postsynaptic inhibition appears to play some role in altering the degree of damage of the second order neurons postsynaptic to the peripherally axotomized primary neurons. This would suggest that the strychnine-enhanced transneuronal destruction of dorsal horn neurons following peripheral nerve injuries was mainly due to the excessive excitation of second order neurons. As for the source of such excessive excitation of dorsal horn neurons, injured primary neurons innervating the stump neuroma have been known to show ectopic electrical discharge [10,13,14,20,28,29]. If abnormal discharge of injured primary neurons was the direct cause of transneuronal destruction of dorsal horn neurons, the injury discharge, which immediately follows the peripheral nerve injury, might also be related to the strychnine-enhanced transneuronal destruction of second order neurons. In addition, administration of chemical convulsants other than strychnine would also enhance the transneuronal destruction if they removed either pre- or postsynaptic inhibition of second order neurons. Therefore, we examined acute effects of transection of the inferior alveolar nerve under the influence of the convulsants strychnine, picrotoxin or bicuculline. Transection was repeated 7 times to exaggerate the injury discharge.

Materials and methods A total of 50 rats (body weight 200 g) of the Sprague-Dawley strain were used. Fourteen animals underwent unilateral transection of the inferior alveolar nerve under the influence of strychnine (1 mg/kg), picrotoxin (0.5 mg/kg) or bicuculline (2 mg/kg). All the drugs were dissolved in saline. Surgical procedures were performed under anesthesia with ethyl carbamate (1.3 g/kg, i.p.). The buccal skin was incised, masseter muscle was retracted, and a small amount of bone was removed with the aid of a dental burr to open the mandibular canal. A 5 mm segment of the inferior alveolar nerve was exposed about 1 mm distally to the mandibular foramen. Starting at the distal end and moving in the proximal direction the nerve was repeatedly transected 7 times at intervals of 1 min with the aid of iridectomy scissors. At the end of surgery the muscle and skin were sutured in 2 layers. All drugs were administered i.p. 1 min before the initial transection. In addition to the above, 13 animals received single injections of drugs 1 min after but not before the series of transections, 14 animals received only drug injections under anesthesia without nerve transection, and 5 animals received only the nerve transections. Furthermore, 4 animals were anesthetized with ethyl carbamate but did not

387

undergo further experimental manipulations. These 4 animals served for evaluation of the adequacy of the following histological procedures. Approximately 18 h after the above surgery or administration of drugs, animals were anesthetized with ether and transcardially perfused with 1% glutaraldehyde, 1% formaldehyde and 0.05 mM CaCl, in 0.12 M phosphate buffer (pH 7.3). Thirty minutes after completion of the perfusion, the brain-stem was removed and stored in a fresh fixative for another 30 min at room temperature. The brain-stem was osmicated in a phosphate-buffered 2% 0~0, solution, dehydrated in graded alcohols and embedded in an epoxy resin. The tissue blocks were so trimmed that sectioning was started at the rostralmost level of the medullary dorsal horn (within 1 mm caudal to the obex) and proceeded caudally. Transverse 1 pm sections of the medullary dorsal horn (less than 1 mm caudal to the obex) were cut, stained with 1% toluidine blue, dissolved in aqueous solution of 1% sodium borate and examined under a light microscope. From the rostralmost 5-6 serial sections which covered the entire transverse section area of the medullary dorsal horn ipsilateral to neurotomy without defect or knife mark, a representative section was selected for each animal and the number of unequivocally identified dark neurons (see below) in laminae I-IV of the medullary dorsal horn ipsilateral to the nerve transection were counted (see Sugimoto et al. [27] for details). All the cell counting was performed by a single examiner who did not participate in the histological processing including sectioning and was not informed about the experimental condition of the animals from which the sections were obtained.

Results The 1 pm sections of the medullary dorsal horn ipsilateral to the nerve transection revealed many neurons, which stained deeply with toluidine blue, in all animals pretreated with any of the 3 drugs (Figs. 1 and 2). These neurons resembled the damaged neurons which were found in the medullary dorsal horn following chronic transection of the inferior alveolar nerve and subsequent strychnine administration [25] and will be called dark neurons because of their chromophilia. Examples of dark neurons produced by pretreatment with the 3 drugs are shown in Fig. la-c. Their nuclei had a ruffled contour with many shallow indentations, and the nucleoplasm had lost heterochromatin and stained homogeneously dark except for the nucleolus. The cytoplasm of these neurons appeared more deeply stained than that of healthier neurons. In this study, only those neurons, which showed increased stainability of the nucleoplasm, were unequivocally judged to be dark neurons and their number was counted [27]. These dark neurons were mostly found in the dorsal half of the medullary dorsal horn (an example with picrotoxin pretreatment is shown in Fig. 2) which has been shown to receive a dense innervation of primary neurons constituting the ipsilateral inferior alveolar nerve [12,25]. Dark neurons were relatively rare in the ventral half of medullary dorsal horn. Among the 3 drugs examined, picrotoxin produced more dark neurons than strychnine or bicuculline: picrotoxin produced 46 + 13 dark neurons per section

Fig. 1. Examples of dark neurons in the medullary dorsal horn ipsilateral to the nerve transection in toluidine blue stained 1 pm sections. Animals were pretreated 1 min prior to the nerve transection with 1 mg/kg of strychnine (a), 0.5 mg/kg of picrotoxin (b) and 2 mg/kg of bicuculline (c). x 1530.

I. .

Fig. 2. A camera lucida drawing of a single toluidine blue stained 1 nm section of the medullary dorsal horn ipsilateral to the inferior alveolar nerve transection. Broken line indicates the medial border of lamina IV. Each dot represents an unequivocally identified dark neuron. Most of the dark neurons are found in the dorsal half of the medullary dorsal horn across laminae I-IV. This animal received a single injection of picrotoxin (0.5 mg/kg, i.p.) 1 min prior to the nerve transection. x42.

389 TABLE

I

NUMBER DORSAL MENTAL

OF DARK NEURONS PER SECTION MORN IPSILATERAL TO THE NERVE PROCEDURE

For control

experiments

without

transection

(drug only), dnps were counted

Experiment

Number

Cut only

5

Strychnine

Picrotoxin

of animals

on the left side. Mean f S.D. 7.2f

4.4

only

4

pre post

4 5

3.35” 5.6 17.0+ 2.9 * 7.8* 7.0

only

5 5 4

4.2& 5.6 45.8 * 13.3 + * 9.0& 8.8

5 5 4

0.45 0.8 19.X 5 5.2 * * 3.8i 6.5

pre post Ikuculline

(dnps) OF LAMINAE I-IV OF MEDULLARY TRANSECTION FOLLOWING EACH EXPERI-

only pre post

* Values were sjgnjficantl~ greater than cut only (P < 0.05) and drug only (P < 0.07); * * values were significantly greater than cut only f P < 0.01), drug only f P < 0.01) and drug post ( P < 0.01).

(dnps) of laminae I-IV ipsilateral to the nerve transection, while strychnine and bicuc.ulline produced 17 t_ 3 and 20 F 5 dnps, respectively (Table I). However, a qualitative difference was not discernible between these drugs in terms of topographic distribution of dark neurons. The experiments which examined the effects of transection alone, drug administration alone and drug administration 1 min after the final transection, revealed much fewer dnps than those which examined the effects of preadministration of the drugs (Table I). Most of these dark neurons. unlike those seen after preadministration of the drugs, were evenly scattered in the entire dorso-ventral extent of medullary dorsal horn. Even in the experiments in which the inferiar alveolar nerve was transected after or without drug administration, dark neurons did not show a clear topographic distribution pattern which may be related to the nerve transection. In the animals which had undergone neither transection nor drug administration, no dark neurons were found in the medullary dorsal horn.

Discussion The dark neurons observed in this study have to be distinguished from the so-called ‘dark neurons’ which can be seen in autopsy and biopsy materials. The latter is thought to be a fixation artefact (or perhaps a post-mortem change) caused by immersion fixation and can be almost completely eliminated by perfusion fixation which is now routinely used in most histological studies 14,311. The control experiment, in which both surgical manipulations and drug administration were

390

omitted, clearly showed that the artefactual production of ‘dark neurons’ was prevented by the histological procedures in this study. The histological procedures employed in this study, including perfusion fixation, were identical to those successfully used in our previous studies [24-271. With these procedures, 7 or 23 days of strychnine administration (1 mg/kg/day) without nerve transection did not yield dark neurons in the medullary dorsal horn [25-271. Chronic transection of the inferior alveolar nerve alone did not produce a dark neuron, either [25]. Nevertheless, several dark neurons were observed in some of the experiments in which the drug administration alone or transection alone was examined. This may be because some medullary dorsal horn neurons are more vulnerable to acute toxicity of the drugs than others. These dark neurons might have been lost from the medullary dorsal horn, or developed tolerance and recovered in the previous studies with a longer period of strychnine administration [25,26]. As to the dark neurons observed following transection alone, they might have undergone transneuronal destruction in response to the acute nerve transection. The topographic distribution of dark neurons produced by the nerve transection might have been obscured by the dark neurons caused by tissue damage due to surgical manipulations such as the skin incision and muscle retraction. Interestingly, however, the studies examining the effect of acute lesioning of the peripheral nervous system (mostly the dorsal rhizotomy) have never shown morphological changes in the dorsal horn neurons [e.g., 181. This might have been due to the differences in transection site (dorsal root vs. peripheral nerve) and transection procedure (single cut vs. 7 times). Furthermore, a pentobarbital anesthetic used in the previous studies might have affected the response of primary and secondary neurons to the acute injuries. The present data provide evidence to support the hypothesis that injury discharges following peripheral nerve lesions may be responsible for the transneuronal changes of second order neurons. Judging from the topographic distribution and the number of dark neurons, preadministration of drugs appears to have enhanced the transneuronal effect of nerve transection. Since the drugs administered 1 min after the nerve transection did not enhance the transneuronal effect, the destructive transneuronal effect must have developed immediately after transection and subsided within 1 min following transection. Many different changes, such as loss of chemical substances [2,19] and ectopic spike generation within the neuroma [10,13,14,20,28,29], are known to take place in injured primary neurons. Latency and duration of these changes, however, were much longer than those of the destructive effect demonstrated in this study. Injury discharge is the only known episode with such a short latency and duration which follows transection of a nerve [30]. Strychnine would certainly enhance the physiological consequence of such ectopic discharge by antagonizing glycinergic postsynaptic inhibition. Picrotoxin and bicuculline would also enhance it by removing presynaptic inhibition mediated by GABA [5,7,8,16,17,22,26]. Since GABAergic axons were shown to be presynaptic to dendrites of dorsal horn neurons as well as to primary afferent terminals [1,15], picrotoxin and bicuculline might also have enhanced the transneuronal effect by removing the postsynaptic inhibition. Although the exact mechanism involved in the

391

neuronal damage in this study is unclear, it is possible that excessive excitation caused physiologically intolerable ionic imbalance across the membrane or metabolic exhaustion of the affected neurons, Excessive excitation of neurons caused by chemical convulsants or electrical stimulation is known to induce degeneration of postsynaptic neurons along seizure pathways [3,6,21,23]. Chronic transneuronal destruction of cell bodies of dorsal horn neurons was seen only when strychnine was administered at certain posttransectional intervals [25-271. This, however, does not mean that nerve transection does not cause transneuronal destruction by itself. Chronic nerve transection did cause transsynaptic destruction at the electron microscopic level [9,24,25] and strychnine only enhanced it to be observable at the light microscopic level [25]. Some neuronal changes at the light microscopic level were also seen as a result of acute nerve transection without drugs, or as a result of drug administration without transection, although neither the nerve transection nor the drug administration alone caused neuronal damage in a degree equal to that caused by preadministration of the drugs in intensity and topographic distribution. Clinical experiences have shown that there is some difference between surgical procedures of peripheral nerve transection for limb amputations (such as cutting with a sharp instrument and crushing) in frequency and duration of posttraumatic sensory disorders. Surgical removal of an amputation neuroma is sometimes effective for the reduction or elimination of sensory disorders. The rapid transneuronal destruction of dorsal horn neurons as shown in this study may underlie the above clinical observations. It may be through this transneuronal destruction that the central nervous system readjusts itself to disconnection from the original receptive field. Success in such readjustment will, to some extent, depend on the amplitude and duration of ectopic afferent barrage at the time of traumatic or surgical transection of peripheral nerves. In addition, failure in or excess of regulation of such readjustment by pre- and postsynaptic inhibitions may lead to posttraumatic sensory disorders such as causalgia and phantom limb. Pharmacological modification of pre- and postsynaptic inhibitions may help to prevent or treat posttraumatic sensory disorders.

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