- Email: [email protected]
Autotomy following limb denervation: Effects of previous exposure to neurectomy
10.5
Pa& 21 (1985) 105-115 Elsevier PA1 00720
Research Reports
Autotomy ~o~~~win~Limb Denervation: Effects of Previous Exposure to ~eurectomy
1
Anatoly C. Rabin and Edmund G. Anderson 2 ~epar~rnen~ of Pharma~olo~,
Uni~ersi~ of Illinois College of Medicine, Chicago, fL 606it
(U.S.A.)
(Received 25 August 1983, accepted 16 October 1984)
Summary Neurectomy, dorsal rhizotomy or spinal cord lesions can evoke self-attack and autotomy of portions of insensate limbs, This behavior has been attributed to dysesthesia and/or pain. However, the majority of animals subjected to these lesions do not exhibit self-mutilation. This study was designed to test for individual predisposition to self-mutilative behaviors by effecting two sequential limb denervations. In susceptible animals, denervation of the first limb induced circulatory, trophic and inflammatory reactions and/or autotomy. Inflammation developed slowly in one or two digits of some animals and then spontaneously resolved. Other animals attacked the tips of inflamed digits effecting local autotomy. Still other animals aggressively attacked multiple digits, rapidly developed diffuse in~ammation and produced extensive autotomy. Denervation of a second limb, following complete healing of all tissue injury, induced by the first denervation, produced several dramatic responses. Nearly 90% of those responding with autotomy to the first denervation responded with autotomy to the second denervation. In contrast, only 11% of the non-attackers showed autotomy following the second denervation. The inflammatory and autotomy responses to the second denervation were markedly reduced in latency and increased in intensity. The pattern of autotomy was stereotyped, initiated at the digit tips, progressed proximally, lead by advancing inflammation. It is suggested that a strong host-response component is involved in the a~totomy response.
’ Supported in part by USPHS Grant DE 5390, ’ Please address alI correspondence to: E.G. Anderson, Ph.D., Department of Pharmacology, University of Illinois College of Medicine, 835 S. Woicott, Chicago, IL 60612, U.S.A. 03043959/85/$03.30
Q 1985 Etsevier Science Publishers B.V. (Biomedical Division)
Introduction Lesions of peripheral nerves [8,19,21}, dorsal roots ~2-4,7,IO] or the spinal curd 19,111can induce the strange behavior of self-mutilation in a variety of species. Some animaIs respond to these lesions by scratching skin wounds over the denervated limb and/or self-amputation of the distal appendages. Others exhibit normal care of the limb. This self-mutilation has been interpreted as a response to dysesthesic referred sensation, or pain, and suggested as an animal model for deafferentat~on pain [6,10,13,20]. However, the normal group behavior, regular weight gain and the lack of overt suffering during the self-mutilation process are inconsistent with the hypothesis of painful sensation. The validity of this hypothesis and the model as representing chronic pain was recently challenged after review of both animal and clinical studies [18]. A puzzling aspect of all studies of autotomy behavior is the large percentage of animals which do not respond with autotomy in spite of identical neural lesions. This study was initiated with the purpose of determining if the response of autotomy, or lack thereof, was an individual specific reaction. Our strategy was to determine the response to the denervation of one limb, and then see if the response remained the same to denervation of a contralateral limb in the opposite quadrant. The results of these experiments indicate that factors other than pain are aIs0 important in evoking the behavior of autotomy.
Methods
This work was carried out on male Sprague-Dawley rats, which were divided into 3 groups. In the first group (36 rats) the right forelimb was denervated first. Ten weeks later, after the results were observed, a denervation of the left hind limb was performed in the same animals. In the second group (25 rats) the limb order was reversed. In animals of the third group (19 rats) both limbs were denervated simultaneously. The animals weighed between 225 and 250 g at the time of the first operation. They were anesthetized with pentobarbital(50 mg/kg), given after atropine methylnitrate (0.05 mg). Denervation of the forelimb was performed by section of the brachial plexus through two approaches; the main part of the plexus was ligated and cut in the subclavicular area. Additionally, sections of the C, and C, spinal nerves were made in the neck. Denervation of the hind limb was carried out in ad-t~gh by ligation and section of the sciatic and saphenous nerves. The animals were housed, two per cage, and postoperative care was given to minimize any disability. It was found, however, that even after denervation of two limbs the animals exhibited no evidence of suffering. They gained weight normally and showed normal group behavior. They were able to ~bulate, to rear and required no speciat care to maintain feeding. Observations were made daily, at the same time. No attempt was made to observe animals in the act of autotomy.
107
However, since the autotomy process usually occupied a period of several days, daily observations allowed a sequential sampling of events. Any animal showing signs of discomfort or obtaining the maximal allowable autotomy score was removed from the study. The characteristics of the neurological deficit, appearance of the denervated limb, the surface temperature of the limb (measured on the palmar surfaces), and the development of inflammation and autotomy were carefully observed and recorded. Temperature recordings were made from manually restrained rats with a small ther~stor probe (0.25 mm diameter, 0.I see time constant). Gross qualitative estimates of the occurrence and type of bleeding were obtained from the color and extent of blood stains on the bedding chips in the cage. Autotomy
score
In contrast to other studies [8,19-211 the scoring procedures included only obvious cases of digit removal. For reasons explained or removed nails and infla~ation of digit tips were not included in point was recorded for the removal of half of each digit. Thus a total of 8 could be reached if all 4 digits of the forepaw were removed. In when all 5 digits were involved the maximal score was 10. Any obtained a maximal score was removed from the study at that point.
for autotomy below, broken the score. One possible score the hind limb, animal which
Results (1) The response of the distaf limb to de~e~at~o~
Daily observations revealed that a sequence of circulatory, trophic and inflammatory reactions in the denervated limb followed denervation. Thermistor recordings from the palmar surfaces showed that immediately following surgery the denervated limbs became warmer. The denervated hind foot averaged l.S’C f SE. 0.23 warmer and the denervated front paw averaged 5.2’C f SE. 0.30 warmer than their unoperated contralateral limbs. This probably results from interruption of sympathetic tone. Twenty-four hours later the denervated hind paws now averaged 25°C f SE. 0.51 cooler, and the denervated front paws were only slightly warmer (0.92”C f SE. 1.03) than their normal counterparts. By 11 days some animals exhibited cooler denervated paws while others had no temperature differences. The measurements became erratic and highly affected by struggle during restraint. However, the data suggest that compensatory processes of skin blood flow occur, and that these reactions are variable. Trophic changes became obvious in many animals within a few days. The nails thickened and darkened. A glossy-like skin with some scaly desquamation appeared at the distal digits. Poor limb positioning and grooming activity resulted. Broken or amputated nails appeared to be secondary to poor limb usage and normal grooming rather than abnormal autotomy behavior. Circulatory and trophic changes were sometimes followed by purulent infections, frequently initiated around the nail bed. In other cases, inflammatory infiltrate
108
involved the whole distal phalanges of one or two digits. Local inflammation was more common in the hind than in the forepaw. In 18 cases following either the first or second denervation, the local inflammatory reaction developed over several days and then subsided spontaneously without evidence of autotomy. In 18 other cases, inflammation was followed by local attack always directed toward inflamed areas. The period between the first noticeable in~ammation and the beginning of autotomy averaged 9.6 it SE 1.88 days. In these cases, the local in~ammation was confined to one or two digits, and chewing did not evoke inflammatory spread. Instead, chewing appeared to remove tissue damaged by inflammation, and local healing followed. This localized chewing progressed slowly over many days and produced modest autotomy scores. In 45% of the chewers, autotomy was not preceded by a noticeable local inflammatory reaction. Overnight these animals initiated aggressive attack toward multiple digits. An immediate inhalator reaction developed in the whole distal limb. The entire paw rapidly became endematous reddened and then cyanotic, Autotomy then progressed rapidly with self-amputation of most digits usually occurring. However, only scanty signs of bleeding were observed, with some dark brown stains appearing on the bedding. These ‘aggressive’ responders differed from ‘local’ responders, described above, by executing higher autotomy scores. The pattern of autotomy was stereotyped. Both local and aggressive chewers began autotomy at the tips of the digits and progressed proximally. This strange pattern contrasted with the pattern of self-biting behavior exhibited by some rats which initiated post-denervation self-attack immediately following recovery from anesthesia. These animals (we have observed 14 cases out of 169 rats) attacked the base of the most medial digit and exhibited significant bleeding leaving many bright red stains on the bedding. They did not produce autotomy if the bleeding was arrested. These animals showed immediate attack on insensate tissue at the most physically convenient site. In contrast, the distal-proximal pattern of delayed autotomy seemed to be a purposefully directed behavior, rather than an indescriminate attack on insensate tissue. The picture emerging from these observations is that limb denervation elicits a loose sequence of reactions. An immediate vasodilation is followed by variable shortand long-term compensatory changes. The degree of compensation appears individualized; ranging from modest circulatory and trophic changes through local inflammatory reactions. Local attack frequently followed local inflammation, and multiple digit attack induced diffuse inflammation of the whole appendage. Two sub-populations
of responders to limb denervation
The incidence, latency, time course and intensity of autotomy observed in this study (Tables I and II) agree with those previously described by many workers. In groups with denervation of either a right forelimb or a left hind limb about one-third of the animals responded with autotomy (groups 1 and 2). When the right forelimb and the left hind limb were denervated simultaneously (group 3) the percentage of rats with autotomy was not increased. The latency of the first attack towards denervated limbs ranged from a few days to 6 weeks. The mean latency to autotomy
109
TABLE
I
THE INCIDENCE
OF AUTOTOMY Denervation one limb
Group 1 Forelimb
hind limb
(1st)
FOLLOWING of
LIMB DENERVATION
Denervation two limbs
of
Autotomy of the second limb among animals With autotomy following 1st denervation
Without autotomy following 1st denervation
n = 34 23%
n = 34 32%
n=S* 87.5%
n=26* 15%
n = 24 25%
n = 24 29%
n=6* 83%
n=18* 6%
(2nd)
Group 2 Hind limb
forelimb (2nd)
(1st) Group 3 Forelimb
n=18 28%
+ hind limb
* P c 0.01, x2 tests.
in rats with denervation of one or two limbs, effected in one surgery, did not differ significantly (Table II). All rats, receiving a single limb denervation, were separated into sub-groups on TABLE
II
LATENCIES
TO AUMTOMY
AND INFLAMMATION
Responders
Group 1 Forelimb (1st)
Double hind limb (2nd)
Autotomy
latency
Double forelimb (2nd) Single
Croup 3 Forelimb
latency
1st denervation
2nd denervation
n-7 28.1 ** + SE. 4.08
n=7 10.3 ** + S.E. 3.59
n=5 23.4 I SE. 3.93
n=5 8.2 f SE. 1.87
23 *
n=4 29.3 f S.E. 5.62
n=S 29.4 *** + SE. 5.73
n=5 6.8 *** + SE. 2.04
24 +
34 * n=6 30.8 * S.E. 3.54
matched
Inflammation
(days)
2nd denervation
+ hind limb
* Single observation. ** P < 0.01, Wilcoxon *** P < 0.05.
LIMB DENERVATION
1st denervation
Single
Group 2 Hind limb (1st)
AFTER
pairs test.
n=14 19.1 f S.E. 3.61 n-5 17.4 It SE. 5.33
5*
n=3 19.7 f S.E. 6.01
110
the basis of whether they exhibited autotomy (chewers) or not (non-chewers). They were then subjected to denervation of the second limb 10 weeks after the first surgery. At that time all self-inflicted wounds had healed and epithelialized. A marked difference in response was observed between chewers and non-chewers to denervation of the second limb (Table I). Eighty-fight percent of the responders to the first denervation in group 1 and 83% of the responders in group 2 exhibited autotomy of the second denervated limb, while only 15% and 6% of the two subgroups of non-responders became responders following the second denervation (Table I). These differences were highly significant (P -C 0.01, x2 test). In addition, nearly 80% of the rats who were ‘aggressive chewers’ following the first denervation exhibited aggressive chewing following the second denervation. Also, both of the aggressive chewers in group 3 (those receiving simultaneous denervation of two limbs) attacked both Iimbs, and each limb exhibited a relatively high autotomy score. The above data indicate that rats in the two sub-groups (responders and non-responders) represent two sub-populations. Furthermore, amongst the responders, aggressive chewers remain aggressive chewers.
intensification of autotomy after sequential limb denervation Another striking observation was that when a second limb denervation was implemented the latency to autotomy amongst the responders was reduced to one-third or one-fourth of its value following the first denervation {Table II). Thus, in group 1 the mean latency was reduced from 28.1 days following denervation of the forelimb to 10.3 days following the hind limb denervation. Similarly, in group 2 the mean latency significantly decreased from 29.4 days following hind limb denervation to 6.8 days following forelimb denervation. Analysis of the time distribution of the latency (Fig. 1) revealed that very few animals, following the first dene~ation, responded with autotomy in the first 2 weeks with the majority responding with a 20-40 day latency. Furthermore, there was a clear cut-off with no animals responding beyond 40 days. In contrast, the vast majority of the animals after the second denervation responded within the first 10 days. Both distributions were strongly skewed, but in opposite directions. Therefore, non-parametric statistics were used for all comparisons. It is important to point out that neither rats with simultaneous denervation of both limbs, nor those with successive denervations, but responding only to denervation of the second limb, exhibited the shortened latency (Table II). Sequential limb denervation also produced a significant increase in the autotomy score. Because of the different number of digits in the front- and hind limb, and therefore the different possible maximal score, the data were transformed as a percent of the maximal score and compared using the Mann-Whitney U test. A significant increase of the autotomy score after a second denorvation was confirmed (P < 0.05). Like the autotomy response, all other ma~festations of the denervation syndrome were intensified by sequential limb denervation. The latency to appearance of local inflammation decreased by over 60% with denervation of the second limb (Table II).
111 NUMBER OF ANIMALS
i-l
DAYS
Fig. 1. The ~s~bution of iatencies to autotomy in rats subjected to sequential denervation of two limbs 10 weeks apart, and responding with autotomy to both denervations. The upper and lower histograms present the distribution of the latencies to autotomy following the second and first denervation, respectively. Abscissa: n = the number of animals. Ordinate: days after the first or second denervation. TABLE III MEAN SCORE OF AUTOTOMY FOLLOWING DENERVATION Responders
Forelimb (1st)
Hind limb (1st)
hind limb (2nd)
forelimb
Sequential denervation Denervated 1st
Denervated 2nd
Double
n=7 5.1 **/max 8
n=l 8.1 **/max 10
Single
5*
5*
Double
n-5 5.6 **/max 10
n=5 6.4 **/max 8
3*
n=4 5.2
(2nd) Single
* Single observation. ** P c 0.05,Men-W~tney
U test.
Simultaneous denervation
n=3 5.7
n=S 7.0
112
Previous exposure to denervation increased the likelihood of an inflammatory response to a second denervation. Of the 22 animals which failed to show any response to denervation of the first limb, 14 showed local inflammation to denervation of the second limb. Four of these proceeded to autotomy. When, however, local inflammation appeared in a previous non-responder, it required as long a latency as those responding to the first denervation. Although the number of rats attacking both limbs is relatively small in all 3 groups, the proportion of ‘double responders’ after sequential denervation of the second limb is higher than after simultaneous denervation of both limbs. In addition, many local responders to the first denervation became aggressive responders following the second denervation. The weight of the above data strongly supports the concept that the sequential procedure intensifies the response to the second denervation. Another observation from the sequential denervation procedure was that there was a correlation between the latencies within each animal of the responses to denervation of the first vs. the second limb. A correlation coefficient was calculated (r = 0.6061, slope = 0.41) which showed a weak correlation, which was just significant (P = 0.05). Discussion The above findings demonstrate that the autotomy response to denervation is individually specific. Animals responding with autotomy to denervation of one limb responded with autotomy to denervation of a second limb. Conversely, non-responders remained non-responders following a second denervation. Responders also revealed a dramatic shortening of the latency to both inflammation and autotomy following a second denervation as well as an intensification of these responses. Non-responders showed an increased incidence of inflammation following a second denervation (as compared to the first denervation), but few developed autotomy. Within individual responders, the duration of the latency to autotomy following the second denervation, though markedly shortened, correlated significantly with the latency following the first denervation. Thus, these data indicate that the mechanisms underlying susceptibility to an intensification of tissue and behavioral responses to limb denervation have a strong host-response component which is consistent within individuals but varies greatly between individuals. Daily observations of the responses to denervation yielded a series of time-lapse pictures of the denervation syndrome which suggest important relationships. Infl~mation u~fo~y started at the tips of one or two digits and spread proximally. Inflammation often resolved spontaneously, but was frequently followed by autotomy. Autotomy also started at the digit tips and progressed proximally, which is not a behavior of convenience. Two types of autotomy were observed. Local chewers uniformly initiated attack on an inflamed digit and over many days chewed adjacent tissue which was also inflamed. These local chewers obtained modest autotomy scores.
113
Aggressive chewers followed a different program. Without previously observable inflammation, multiple digit tips were suddenly attacked. Diffuse inflammation of the distal limb rapidly followed. The inflammatory spread was vigorously pursued by aggressive chewing, resulting in rapidly executed high autotomy scores. The lack of observable signs of signific~t bleeding, even during the most extensive autotomy, suggested that circulatory changes preceded these attacks. The difference in the programming of autotomy between local and aggressive chewers could suggest different mechanisms for these two responses. However, a number of commonalities exist between these two forms of autotomy. There are similar latencies, a distal-proximal progression, lack of evident bleeding, sensitization to both forms of autotomy by a previous autotomy experience. Thus we suspect a common underlying cause which remains unidentified. A possible component of this underling cause may be ischemia. This is suggested by the i~tiation of both inflammation and autotomy at digit tips, where circulation is easily compromized, and the lack of signs of significant bleeding, The visible manifestations of the denervation syndrome in the distal rat limb is strongly reminiscent of the vasomotor and nutritional changes after peripheral nerve injuries in humans. Clinical studies [1,5,12,15,17] demonstrate that division of a mixed peripheral nerve first causes vasodilatation, due to the interruption of the sympathetic innervation. Vascular tone returns in a few days, and 2-3 weeks later the denervated fingers become cooler and often appear cyanotic. The development of late coldness is associated with the loss of sensory cutaneous fibers and the axon reflex [12,17,23]. It is noteworthy that, in humans, the ischemia following peripheral nerve injury is not linked with chronic deafferentation pain [15]. In contrast to the above parallels between rat and human responses to deafferentation, rat autotomy responses differ in some ways from deafferentation pain responses in humans. Deafferentation pain induces acute suffering which often dominates human behavior 1221. In contrast, rats with denervated limbs exhibited normal grooming behavior, weight gain and group activity even during the phase of active autotomy. Such behavior seems inconsistent with chronic pain. In humans, deafferentation pain usually begins immediately after neurotrauma, lasts for years and is exacerbated by stress [16,22]. In rats, autotomy had a 2-3 week latency, a maximal duration of 3 weeks and could not be reactivated in that limb, even under the stress of denervating a second limb. In humans chronic pain is not relieved by amputation. In rats, once self-amputation was completed, no further interest was displayed in the insensate stumps. This suggest that the stimulus which induces autotomy behavior fades during autotomy. One similarity between pain and autotomy is that increasing deafferentation increases pain intensity in humans [22] and the degree of autotomy in rats [13,14,20]. However, the intensification of autotomy following sequential limb denervation cannot be explained by summing the deafferentation produced by the two denervations. Simultaneous denervation of two limbs resulted in the same incidence and intensity of autotomy, as a single denervation of either limb. Thus, the shortened latency and intensified denervation syndrome which occurred when two limbs were denervated sequentially, 10 weeks apart, suggest that some other process is involved in this sensitization.
114
Dennis and Melzack [4] observed that formaldehyde injection into the paw shortened the latency to autotomy. They attributed this to a ‘pain memory,’ but noted that non-neural mechanisms are involved since formaldehyde also reduced the latency when injected after limb deafferentation; a situation that would prevent any pain perception. Our data wouid be readily explained by non-neural mechanisms. We suggest that inflation, and possibly local ischemia, may be components of the non-neural mechanisms. A key question remaining unanswered is, what stimulus directs self-attack. Dysesthesia appears the most likely candidate driving scratching behavior following peripheral neurectomy [13,14]. However, autotomy behavior may involve a combination of neural and humoral signals. Whatever the stimulus, our data suggest that intimation is an important correlate of autotomy behavior. Finally, the observations presented in this study suggest that the entire denervation syndrome which follows peripheral nerve section in rats closely mimics many changes observed in humans and, thus, is an important model in all its manifestations.
References 1 Atlas, L.N., The etiology of vasomotor and nutritional changes following peripheral nerve section, Surgery, 4 (1938) 718-721. 2 Basbaum, A.I., Effects of central lesions on disorders, produced by multiple dorsal rbizotomy in rats, Exp. Neurol., 42 (1974) 490-501. 3 Berman, D. and Rodin, B.E., The influence of housing condition on autotomy following dorsal rhizotomy in rats, Pain, 13 (1982) 307-311. 4 Dennis, S.G. and Melzack, R., Self-mutilation after dorsal rhizotomy in rats: effects of prior pain and pattern of root lesions, Exp. Neurol., 65 (1979) 412-421. 5 Doupe, J., Studies in denervation. B. The circulation in denervated digits, J. Neural. Psychiat.. 6 (1943) 97-111. 6 Duckrow, R.B. and Taub, A., The effect of dipheny~yd~toin on self-mutilation in rats produced by unilateral multiple dorsal rhizotomy, Exp. Neural., 54 (1977) 33-41. 7 Hnik, P., Motor function disturbances and excitability changes following deafferentation, Physiol. bohemoslov, 5 (1956) 305-315. 8 Inbal, R., Devor, M., Tuchendler, 0. and Leiblich, I., Autotomy following nerve injury: genetic factors in the development of chronic pain, Pain, 9 (1980) 327-337. 9 Ingebritsen, O.C., Co-ordinating mechanisms of the spinal cord, Gen. psychol. Monogr.. 13 (1933) 488-555. 10 Levitt, M. and Heybach, J.P., The d~fferenta~on syndrome in geneticalfy blind rats: a model of the painful phantom limb, Pain, 10 (1981) 67-73. 11 Levitt, M. and Levitt, J.H., The deafferentation syndrome in monkeys: dysesthesias of spinai origin, Pain, 10 (1981) 129-147. 12 Lewis, T. and Pickering, G.W., Circulatory changes in the fingers in some diseases of the nervous system, with special reference to the digital atrophy of peripheral nerve lesions, Clin. Sci.. 2 (1936) 149-17s. 13 Lombard, M.-C., Nashold, B.S. and Albe-Fessard, D., Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible animal model of chronic pain, Pain, 6 (1979) 163-174. 14 Rabin, A.G., Anderson, E.G. and Levy, R.A., Autotomy following limb denervation: effects of sparing selected afferents, Pain, 21 (1985) 117-128. 15 Richards, R.L.. Vascular and nutritional changes after peripheral nerve injury in man. In: P.J. vi&en
115
16 17 18 19 20
21
22 23
and G.W. Bruyn (Eds,), Handbook of Clinical Neurology, Vol. 7, North-Hoii~d, Amsterdam, 1970, pp. 265-275. Seddon, H., Surgical Disorders of the PeripheraI Nerves, Churchill-Livingstone, Edinburgh, 1975. Shepherd, J.T., Physiology of the Circulation in Human Limbs in Health and Disease, Saunders, Philadelphia, PA, 1963. Sweet, W.H., Animal models of chronic pain: their possible validation from human experience with posterior rhizotomy and congenital analgesia, Pain, 10 (1981) 275-295. Wall, P.D., Scadding, J.W. and Tomkiewicz, M.M., The production and prevention of experimental anesthesia dolorosa, Pain, 6 (1979) 175-182. Wall, P.D., Devor, M., Inbai, R., &adding, J.W., Schonfeld. D., Zeltzer, Z. and Tomkiewicz, M.M., Autotomy foilowing peripheral nerve lesions: experimental anaesthesia dolorosa, Pain, 7 (1979) 103-113. Wiesenfeid, Z. and Lindblom, U., Behavioral and electrophysiological effects of various types of peripheral nerve lesions in the rat: a comparison of possible models for chronic pain, Pain, 8 (1980) 285-298. Wynn Parry, C.B., Pain in avulsion lesions of the brachial plexus, Pain, 9 (1980) 41-53. Zuckerman, S. and Ruth, T.C., Spinal roots and tracts in the regulation of skin temperature, Amer. J. Physiol., 109 (1934) 116-117.
Pa& 21 (1985) 105-115 Elsevier PA1 00720
Research Reports
Autotomy ~o~~~win~Limb Denervation: Effects of Previous Exposure to ~eurectomy
1
Anatoly C. Rabin and Edmund G. Anderson 2 ~epar~rnen~ of Pharma~olo~,
Uni~ersi~ of Illinois College of Medicine, Chicago, fL 606it
(U.S.A.)
(Received 25 August 1983, accepted 16 October 1984)
Summary Neurectomy, dorsal rhizotomy or spinal cord lesions can evoke self-attack and autotomy of portions of insensate limbs, This behavior has been attributed to dysesthesia and/or pain. However, the majority of animals subjected to these lesions do not exhibit self-mutilation. This study was designed to test for individual predisposition to self-mutilative behaviors by effecting two sequential limb denervations. In susceptible animals, denervation of the first limb induced circulatory, trophic and inflammatory reactions and/or autotomy. Inflammation developed slowly in one or two digits of some animals and then spontaneously resolved. Other animals attacked the tips of inflamed digits effecting local autotomy. Still other animals aggressively attacked multiple digits, rapidly developed diffuse in~ammation and produced extensive autotomy. Denervation of a second limb, following complete healing of all tissue injury, induced by the first denervation, produced several dramatic responses. Nearly 90% of those responding with autotomy to the first denervation responded with autotomy to the second denervation. In contrast, only 11% of the non-attackers showed autotomy following the second denervation. The inflammatory and autotomy responses to the second denervation were markedly reduced in latency and increased in intensity. The pattern of autotomy was stereotyped, initiated at the digit tips, progressed proximally, lead by advancing inflammation. It is suggested that a strong host-response component is involved in the a~totomy response.
’ Supported in part by USPHS Grant DE 5390, ’ Please address alI correspondence to: E.G. Anderson, Ph.D., Department of Pharmacology, University of Illinois College of Medicine, 835 S. Woicott, Chicago, IL 60612, U.S.A. 03043959/85/$03.30
Q 1985 Etsevier Science Publishers B.V. (Biomedical Division)
Introduction Lesions of peripheral nerves [8,19,21}, dorsal roots ~2-4,7,IO] or the spinal curd 19,111can induce the strange behavior of self-mutilation in a variety of species. Some animaIs respond to these lesions by scratching skin wounds over the denervated limb and/or self-amputation of the distal appendages. Others exhibit normal care of the limb. This self-mutilation has been interpreted as a response to dysesthesic referred sensation, or pain, and suggested as an animal model for deafferentat~on pain [6,10,13,20]. However, the normal group behavior, regular weight gain and the lack of overt suffering during the self-mutilation process are inconsistent with the hypothesis of painful sensation. The validity of this hypothesis and the model as representing chronic pain was recently challenged after review of both animal and clinical studies [18]. A puzzling aspect of all studies of autotomy behavior is the large percentage of animals which do not respond with autotomy in spite of identical neural lesions. This study was initiated with the purpose of determining if the response of autotomy, or lack thereof, was an individual specific reaction. Our strategy was to determine the response to the denervation of one limb, and then see if the response remained the same to denervation of a contralateral limb in the opposite quadrant. The results of these experiments indicate that factors other than pain are aIs0 important in evoking the behavior of autotomy.
Methods
This work was carried out on male Sprague-Dawley rats, which were divided into 3 groups. In the first group (36 rats) the right forelimb was denervated first. Ten weeks later, after the results were observed, a denervation of the left hind limb was performed in the same animals. In the second group (25 rats) the limb order was reversed. In animals of the third group (19 rats) both limbs were denervated simultaneously. The animals weighed between 225 and 250 g at the time of the first operation. They were anesthetized with pentobarbital(50 mg/kg), given after atropine methylnitrate (0.05 mg). Denervation of the forelimb was performed by section of the brachial plexus through two approaches; the main part of the plexus was ligated and cut in the subclavicular area. Additionally, sections of the C, and C, spinal nerves were made in the neck. Denervation of the hind limb was carried out in ad-t~gh by ligation and section of the sciatic and saphenous nerves. The animals were housed, two per cage, and postoperative care was given to minimize any disability. It was found, however, that even after denervation of two limbs the animals exhibited no evidence of suffering. They gained weight normally and showed normal group behavior. They were able to ~bulate, to rear and required no speciat care to maintain feeding. Observations were made daily, at the same time. No attempt was made to observe animals in the act of autotomy.
107
However, since the autotomy process usually occupied a period of several days, daily observations allowed a sequential sampling of events. Any animal showing signs of discomfort or obtaining the maximal allowable autotomy score was removed from the study. The characteristics of the neurological deficit, appearance of the denervated limb, the surface temperature of the limb (measured on the palmar surfaces), and the development of inflammation and autotomy were carefully observed and recorded. Temperature recordings were made from manually restrained rats with a small ther~stor probe (0.25 mm diameter, 0.I see time constant). Gross qualitative estimates of the occurrence and type of bleeding were obtained from the color and extent of blood stains on the bedding chips in the cage. Autotomy
score
In contrast to other studies [8,19-211 the scoring procedures included only obvious cases of digit removal. For reasons explained or removed nails and infla~ation of digit tips were not included in point was recorded for the removal of half of each digit. Thus a total of 8 could be reached if all 4 digits of the forepaw were removed. In when all 5 digits were involved the maximal score was 10. Any obtained a maximal score was removed from the study at that point.
for autotomy below, broken the score. One possible score the hind limb, animal which
Results (1) The response of the distaf limb to de~e~at~o~
Daily observations revealed that a sequence of circulatory, trophic and inflammatory reactions in the denervated limb followed denervation. Thermistor recordings from the palmar surfaces showed that immediately following surgery the denervated limbs became warmer. The denervated hind foot averaged l.S’C f SE. 0.23 warmer and the denervated front paw averaged 5.2’C f SE. 0.30 warmer than their unoperated contralateral limbs. This probably results from interruption of sympathetic tone. Twenty-four hours later the denervated hind paws now averaged 25°C f SE. 0.51 cooler, and the denervated front paws were only slightly warmer (0.92”C f SE. 1.03) than their normal counterparts. By 11 days some animals exhibited cooler denervated paws while others had no temperature differences. The measurements became erratic and highly affected by struggle during restraint. However, the data suggest that compensatory processes of skin blood flow occur, and that these reactions are variable. Trophic changes became obvious in many animals within a few days. The nails thickened and darkened. A glossy-like skin with some scaly desquamation appeared at the distal digits. Poor limb positioning and grooming activity resulted. Broken or amputated nails appeared to be secondary to poor limb usage and normal grooming rather than abnormal autotomy behavior. Circulatory and trophic changes were sometimes followed by purulent infections, frequently initiated around the nail bed. In other cases, inflammatory infiltrate
108
involved the whole distal phalanges of one or two digits. Local inflammation was more common in the hind than in the forepaw. In 18 cases following either the first or second denervation, the local inflammatory reaction developed over several days and then subsided spontaneously without evidence of autotomy. In 18 other cases, inflammation was followed by local attack always directed toward inflamed areas. The period between the first noticeable in~ammation and the beginning of autotomy averaged 9.6 it SE 1.88 days. In these cases, the local in~ammation was confined to one or two digits, and chewing did not evoke inflammatory spread. Instead, chewing appeared to remove tissue damaged by inflammation, and local healing followed. This localized chewing progressed slowly over many days and produced modest autotomy scores. In 45% of the chewers, autotomy was not preceded by a noticeable local inflammatory reaction. Overnight these animals initiated aggressive attack toward multiple digits. An immediate inhalator reaction developed in the whole distal limb. The entire paw rapidly became endematous reddened and then cyanotic, Autotomy then progressed rapidly with self-amputation of most digits usually occurring. However, only scanty signs of bleeding were observed, with some dark brown stains appearing on the bedding. These ‘aggressive’ responders differed from ‘local’ responders, described above, by executing higher autotomy scores. The pattern of autotomy was stereotyped. Both local and aggressive chewers began autotomy at the tips of the digits and progressed proximally. This strange pattern contrasted with the pattern of self-biting behavior exhibited by some rats which initiated post-denervation self-attack immediately following recovery from anesthesia. These animals (we have observed 14 cases out of 169 rats) attacked the base of the most medial digit and exhibited significant bleeding leaving many bright red stains on the bedding. They did not produce autotomy if the bleeding was arrested. These animals showed immediate attack on insensate tissue at the most physically convenient site. In contrast, the distal-proximal pattern of delayed autotomy seemed to be a purposefully directed behavior, rather than an indescriminate attack on insensate tissue. The picture emerging from these observations is that limb denervation elicits a loose sequence of reactions. An immediate vasodilation is followed by variable shortand long-term compensatory changes. The degree of compensation appears individualized; ranging from modest circulatory and trophic changes through local inflammatory reactions. Local attack frequently followed local inflammation, and multiple digit attack induced diffuse inflammation of the whole appendage. Two sub-populations
of responders to limb denervation
The incidence, latency, time course and intensity of autotomy observed in this study (Tables I and II) agree with those previously described by many workers. In groups with denervation of either a right forelimb or a left hind limb about one-third of the animals responded with autotomy (groups 1 and 2). When the right forelimb and the left hind limb were denervated simultaneously (group 3) the percentage of rats with autotomy was not increased. The latency of the first attack towards denervated limbs ranged from a few days to 6 weeks. The mean latency to autotomy
109
TABLE
I
THE INCIDENCE
OF AUTOTOMY Denervation one limb
Group 1 Forelimb
hind limb
(1st)
FOLLOWING of
LIMB DENERVATION
Denervation two limbs
of
Autotomy of the second limb among animals With autotomy following 1st denervation
Without autotomy following 1st denervation
n = 34 23%
n = 34 32%
n=S* 87.5%
n=26* 15%
n = 24 25%
n = 24 29%
n=6* 83%
n=18* 6%
(2nd)
Group 2 Hind limb
forelimb (2nd)
(1st) Group 3 Forelimb
n=18 28%
+ hind limb
* P c 0.01, x2 tests.
in rats with denervation of one or two limbs, effected in one surgery, did not differ significantly (Table II). All rats, receiving a single limb denervation, were separated into sub-groups on TABLE
II
LATENCIES
TO AUMTOMY
AND INFLAMMATION
Responders
Group 1 Forelimb (1st)
Double hind limb (2nd)
Autotomy
latency
Double forelimb (2nd) Single
Croup 3 Forelimb
latency
1st denervation
2nd denervation
n-7 28.1 ** + SE. 4.08
n=7 10.3 ** + S.E. 3.59
n=5 23.4 I SE. 3.93
n=5 8.2 f SE. 1.87
23 *
n=4 29.3 f S.E. 5.62
n=S 29.4 *** + SE. 5.73
n=5 6.8 *** + SE. 2.04
24 +
34 * n=6 30.8 * S.E. 3.54
matched
Inflammation
(days)
2nd denervation
+ hind limb
* Single observation. ** P < 0.01, Wilcoxon *** P < 0.05.
LIMB DENERVATION
1st denervation
Single
Group 2 Hind limb (1st)
AFTER
pairs test.
n=14 19.1 f S.E. 3.61 n-5 17.4 It SE. 5.33
5*
n=3 19.7 f S.E. 6.01
110
the basis of whether they exhibited autotomy (chewers) or not (non-chewers). They were then subjected to denervation of the second limb 10 weeks after the first surgery. At that time all self-inflicted wounds had healed and epithelialized. A marked difference in response was observed between chewers and non-chewers to denervation of the second limb (Table I). Eighty-fight percent of the responders to the first denervation in group 1 and 83% of the responders in group 2 exhibited autotomy of the second denervated limb, while only 15% and 6% of the two subgroups of non-responders became responders following the second denervation (Table I). These differences were highly significant (P -C 0.01, x2 test). In addition, nearly 80% of the rats who were ‘aggressive chewers’ following the first denervation exhibited aggressive chewing following the second denervation. Also, both of the aggressive chewers in group 3 (those receiving simultaneous denervation of two limbs) attacked both Iimbs, and each limb exhibited a relatively high autotomy score. The above data indicate that rats in the two sub-groups (responders and non-responders) represent two sub-populations. Furthermore, amongst the responders, aggressive chewers remain aggressive chewers.
intensification of autotomy after sequential limb denervation Another striking observation was that when a second limb denervation was implemented the latency to autotomy amongst the responders was reduced to one-third or one-fourth of its value following the first denervation {Table II). Thus, in group 1 the mean latency was reduced from 28.1 days following denervation of the forelimb to 10.3 days following the hind limb denervation. Similarly, in group 2 the mean latency significantly decreased from 29.4 days following hind limb denervation to 6.8 days following forelimb denervation. Analysis of the time distribution of the latency (Fig. 1) revealed that very few animals, following the first dene~ation, responded with autotomy in the first 2 weeks with the majority responding with a 20-40 day latency. Furthermore, there was a clear cut-off with no animals responding beyond 40 days. In contrast, the vast majority of the animals after the second denervation responded within the first 10 days. Both distributions were strongly skewed, but in opposite directions. Therefore, non-parametric statistics were used for all comparisons. It is important to point out that neither rats with simultaneous denervation of both limbs, nor those with successive denervations, but responding only to denervation of the second limb, exhibited the shortened latency (Table II). Sequential limb denervation also produced a significant increase in the autotomy score. Because of the different number of digits in the front- and hind limb, and therefore the different possible maximal score, the data were transformed as a percent of the maximal score and compared using the Mann-Whitney U test. A significant increase of the autotomy score after a second denorvation was confirmed (P < 0.05). Like the autotomy response, all other ma~festations of the denervation syndrome were intensified by sequential limb denervation. The latency to appearance of local inflammation decreased by over 60% with denervation of the second limb (Table II).
111 NUMBER OF ANIMALS
i-l
DAYS
Fig. 1. The ~s~bution of iatencies to autotomy in rats subjected to sequential denervation of two limbs 10 weeks apart, and responding with autotomy to both denervations. The upper and lower histograms present the distribution of the latencies to autotomy following the second and first denervation, respectively. Abscissa: n = the number of animals. Ordinate: days after the first or second denervation. TABLE III MEAN SCORE OF AUTOTOMY FOLLOWING DENERVATION Responders
Forelimb (1st)
Hind limb (1st)
hind limb (2nd)
forelimb
Sequential denervation Denervated 1st
Denervated 2nd
Double
n=7 5.1 **/max 8
n=l 8.1 **/max 10
Single
5*
5*
Double
n-5 5.6 **/max 10
n=5 6.4 **/max 8
3*
n=4 5.2
(2nd) Single
* Single observation. ** P c 0.05,Men-W~tney
U test.
Simultaneous denervation
n=3 5.7
n=S 7.0
112
Previous exposure to denervation increased the likelihood of an inflammatory response to a second denervation. Of the 22 animals which failed to show any response to denervation of the first limb, 14 showed local inflammation to denervation of the second limb. Four of these proceeded to autotomy. When, however, local inflammation appeared in a previous non-responder, it required as long a latency as those responding to the first denervation. Although the number of rats attacking both limbs is relatively small in all 3 groups, the proportion of ‘double responders’ after sequential denervation of the second limb is higher than after simultaneous denervation of both limbs. In addition, many local responders to the first denervation became aggressive responders following the second denervation. The weight of the above data strongly supports the concept that the sequential procedure intensifies the response to the second denervation. Another observation from the sequential denervation procedure was that there was a correlation between the latencies within each animal of the responses to denervation of the first vs. the second limb. A correlation coefficient was calculated (r = 0.6061, slope = 0.41) which showed a weak correlation, which was just significant (P = 0.05). Discussion The above findings demonstrate that the autotomy response to denervation is individually specific. Animals responding with autotomy to denervation of one limb responded with autotomy to denervation of a second limb. Conversely, non-responders remained non-responders following a second denervation. Responders also revealed a dramatic shortening of the latency to both inflammation and autotomy following a second denervation as well as an intensification of these responses. Non-responders showed an increased incidence of inflammation following a second denervation (as compared to the first denervation), but few developed autotomy. Within individual responders, the duration of the latency to autotomy following the second denervation, though markedly shortened, correlated significantly with the latency following the first denervation. Thus, these data indicate that the mechanisms underlying susceptibility to an intensification of tissue and behavioral responses to limb denervation have a strong host-response component which is consistent within individuals but varies greatly between individuals. Daily observations of the responses to denervation yielded a series of time-lapse pictures of the denervation syndrome which suggest important relationships. Infl~mation u~fo~y started at the tips of one or two digits and spread proximally. Inflammation often resolved spontaneously, but was frequently followed by autotomy. Autotomy also started at the digit tips and progressed proximally, which is not a behavior of convenience. Two types of autotomy were observed. Local chewers uniformly initiated attack on an inflamed digit and over many days chewed adjacent tissue which was also inflamed. These local chewers obtained modest autotomy scores.
113
Aggressive chewers followed a different program. Without previously observable inflammation, multiple digit tips were suddenly attacked. Diffuse inflammation of the distal limb rapidly followed. The inflammatory spread was vigorously pursued by aggressive chewing, resulting in rapidly executed high autotomy scores. The lack of observable signs of signific~t bleeding, even during the most extensive autotomy, suggested that circulatory changes preceded these attacks. The difference in the programming of autotomy between local and aggressive chewers could suggest different mechanisms for these two responses. However, a number of commonalities exist between these two forms of autotomy. There are similar latencies, a distal-proximal progression, lack of evident bleeding, sensitization to both forms of autotomy by a previous autotomy experience. Thus we suspect a common underlying cause which remains unidentified. A possible component of this underling cause may be ischemia. This is suggested by the i~tiation of both inflammation and autotomy at digit tips, where circulation is easily compromized, and the lack of signs of significant bleeding, The visible manifestations of the denervation syndrome in the distal rat limb is strongly reminiscent of the vasomotor and nutritional changes after peripheral nerve injuries in humans. Clinical studies [1,5,12,15,17] demonstrate that division of a mixed peripheral nerve first causes vasodilatation, due to the interruption of the sympathetic innervation. Vascular tone returns in a few days, and 2-3 weeks later the denervated fingers become cooler and often appear cyanotic. The development of late coldness is associated with the loss of sensory cutaneous fibers and the axon reflex [12,17,23]. It is noteworthy that, in humans, the ischemia following peripheral nerve injury is not linked with chronic deafferentation pain [15]. In contrast to the above parallels between rat and human responses to deafferentation, rat autotomy responses differ in some ways from deafferentation pain responses in humans. Deafferentation pain induces acute suffering which often dominates human behavior 1221. In contrast, rats with denervated limbs exhibited normal grooming behavior, weight gain and group activity even during the phase of active autotomy. Such behavior seems inconsistent with chronic pain. In humans, deafferentation pain usually begins immediately after neurotrauma, lasts for years and is exacerbated by stress [16,22]. In rats, autotomy had a 2-3 week latency, a maximal duration of 3 weeks and could not be reactivated in that limb, even under the stress of denervating a second limb. In humans chronic pain is not relieved by amputation. In rats, once self-amputation was completed, no further interest was displayed in the insensate stumps. This suggest that the stimulus which induces autotomy behavior fades during autotomy. One similarity between pain and autotomy is that increasing deafferentation increases pain intensity in humans [22] and the degree of autotomy in rats [13,14,20]. However, the intensification of autotomy following sequential limb denervation cannot be explained by summing the deafferentation produced by the two denervations. Simultaneous denervation of two limbs resulted in the same incidence and intensity of autotomy, as a single denervation of either limb. Thus, the shortened latency and intensified denervation syndrome which occurred when two limbs were denervated sequentially, 10 weeks apart, suggest that some other process is involved in this sensitization.
114
Dennis and Melzack [4] observed that formaldehyde injection into the paw shortened the latency to autotomy. They attributed this to a ‘pain memory,’ but noted that non-neural mechanisms are involved since formaldehyde also reduced the latency when injected after limb deafferentation; a situation that would prevent any pain perception. Our data wouid be readily explained by non-neural mechanisms. We suggest that inflation, and possibly local ischemia, may be components of the non-neural mechanisms. A key question remaining unanswered is, what stimulus directs self-attack. Dysesthesia appears the most likely candidate driving scratching behavior following peripheral neurectomy [13,14]. However, autotomy behavior may involve a combination of neural and humoral signals. Whatever the stimulus, our data suggest that intimation is an important correlate of autotomy behavior. Finally, the observations presented in this study suggest that the entire denervation syndrome which follows peripheral nerve section in rats closely mimics many changes observed in humans and, thus, is an important model in all its manifestations.
References 1 Atlas, L.N., The etiology of vasomotor and nutritional changes following peripheral nerve section, Surgery, 4 (1938) 718-721. 2 Basbaum, A.I., Effects of central lesions on disorders, produced by multiple dorsal rbizotomy in rats, Exp. Neurol., 42 (1974) 490-501. 3 Berman, D. and Rodin, B.E., The influence of housing condition on autotomy following dorsal rhizotomy in rats, Pain, 13 (1982) 307-311. 4 Dennis, S.G. and Melzack, R., Self-mutilation after dorsal rhizotomy in rats: effects of prior pain and pattern of root lesions, Exp. Neurol., 65 (1979) 412-421. 5 Doupe, J., Studies in denervation. B. The circulation in denervated digits, J. Neural. Psychiat.. 6 (1943) 97-111. 6 Duckrow, R.B. and Taub, A., The effect of dipheny~yd~toin on self-mutilation in rats produced by unilateral multiple dorsal rhizotomy, Exp. Neural., 54 (1977) 33-41. 7 Hnik, P., Motor function disturbances and excitability changes following deafferentation, Physiol. bohemoslov, 5 (1956) 305-315. 8 Inbal, R., Devor, M., Tuchendler, 0. and Leiblich, I., Autotomy following nerve injury: genetic factors in the development of chronic pain, Pain, 9 (1980) 327-337. 9 Ingebritsen, O.C., Co-ordinating mechanisms of the spinal cord, Gen. psychol. Monogr.. 13 (1933) 488-555. 10 Levitt, M. and Heybach, J.P., The d~fferenta~on syndrome in geneticalfy blind rats: a model of the painful phantom limb, Pain, 10 (1981) 67-73. 11 Levitt, M. and Levitt, J.H., The deafferentation syndrome in monkeys: dysesthesias of spinai origin, Pain, 10 (1981) 129-147. 12 Lewis, T. and Pickering, G.W., Circulatory changes in the fingers in some diseases of the nervous system, with special reference to the digital atrophy of peripheral nerve lesions, Clin. Sci.. 2 (1936) 149-17s. 13 Lombard, M.-C., Nashold, B.S. and Albe-Fessard, D., Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible animal model of chronic pain, Pain, 6 (1979) 163-174. 14 Rabin, A.G., Anderson, E.G. and Levy, R.A., Autotomy following limb denervation: effects of sparing selected afferents, Pain, 21 (1985) 117-128. 15 Richards, R.L.. Vascular and nutritional changes after peripheral nerve injury in man. In: P.J. vi&en
115
16 17 18 19 20
21
22 23
and G.W. Bruyn (Eds,), Handbook of Clinical Neurology, Vol. 7, North-Hoii~d, Amsterdam, 1970, pp. 265-275. Seddon, H., Surgical Disorders of the PeripheraI Nerves, Churchill-Livingstone, Edinburgh, 1975. Shepherd, J.T., Physiology of the Circulation in Human Limbs in Health and Disease, Saunders, Philadelphia, PA, 1963. Sweet, W.H., Animal models of chronic pain: their possible validation from human experience with posterior rhizotomy and congenital analgesia, Pain, 10 (1981) 275-295. Wall, P.D., Scadding, J.W. and Tomkiewicz, M.M., The production and prevention of experimental anesthesia dolorosa, Pain, 6 (1979) 175-182. Wall, P.D., Devor, M., Inbai, R., &adding, J.W., Schonfeld. D., Zeltzer, Z. and Tomkiewicz, M.M., Autotomy foilowing peripheral nerve lesions: experimental anaesthesia dolorosa, Pain, 7 (1979) 103-113. Wiesenfeid, Z. and Lindblom, U., Behavioral and electrophysiological effects of various types of peripheral nerve lesions in the rat: a comparison of possible models for chronic pain, Pain, 8 (1980) 285-298. Wynn Parry, C.B., Pain in avulsion lesions of the brachial plexus, Pain, 9 (1980) 41-53. Zuckerman, S. and Ruth, T.C., Spinal roots and tracts in the regulation of skin temperature, Amer. J. Physiol., 109 (1934) 116-117.