Analgesia produced by lidocaine microinjection into the dentate gyms

49(1992) 105-112 0 1992 Eisevier Science Publishers BV. Ail rights reserved 0304-3959/92/$05.00

105

Puin,

PAIN 02004

Analgesia produced by lidocaine microinjection into the dentate gyrus John E. McKenna Department

of Psychology,

McCifl

and Ronald

Melzack

U~it~ersi~, ~o~treol,

Quebec (&nodal

(Received 5 February 1991, revision received 12 September 1991, accepted 19 September 1991)

The local anesthetic lidocaine was injected into the dentate gyrus (DG) of alert, unrestrained rats Summary 10 min prior to investigation within the formalin test. Region& anesthesia of the DG resulted in a reduction of pain scores when administered contralateral to the site of subcutaneous formaIin injection. The analgesic effect was evident 30-50 min after central infusion. These results provide evidence of the involvement of the hippocampal formation (HF) in pain perception. Key words: Dentate gyrus; Hippocampus;

Tonic pain; Local anesthetic; Formalin test

Introduction The hippocampal formation (HF) is an important component of the limbic system (Papez 1937; MacLean 1949) and is assumed to play a role in the affectivemotivational component of pain (Melzack and Casey 1968; Henke 1982). However, there is no conclusive evidence that lesions of the HF, or modulation of hippocampal neural activity, result in a corresponding modulation of pain. It has previously been demonstrated that the injection of a local anesthetic into a brain structure can cause interruption of electrophysiological activity within a discrete area, thus blocking neural activity temporarily (Albert and Richmond 1976; Albert and Madryga 1981; Connors et al. 1982; Sandkuhler and Gebhart 1984; Sandkuhler et al. 1987). It has aIso been shown in our laboratory that when this technique is used to temporarily deactivate limbic structures such as the lateral hypothalamus (Tasker et al. 1987) or cingulum bundle (Vaccarino and Melzack 1989a), significant analgesia may resuh during the late, tonic pain phase of the formaiin test. Physiological (Segal et al. 1972; Sinnamon and Schwartzbaum 1973; Dutar et al. 1985; Khanna and Sinclair 1989), pharmacological (Soulairac et al. 1967; Quirion et al. 1983; Dutar et al. 1985; Gall 1986;

Correspondence to: John McKenna, Department of Psychology, McGill University, 120.5 Dr. Penfield Ave., Montreal, Quebec H3A lB1, Canada.

Sinclair and Lo 1986) and behavioral (Delgado 1955; Halgren et al. 1978; Gloor et al. 1981; Corkin 1984) data suggest that the HF may be involved in pain-related processes. This experiment was therefore undertaken to investigate the possibihty that interruption of neuronal activity in the HF might produce analgesia. The extensive size of the HF precludes anesthetization of the entire structure using a microinjection procedure. Since the anterodorsal region of the hippocampus is believed to be involved in the modulation of responses to motivating stimuii (Nadel 1968; Sinnamon and Schwartzbaum 1973; Sinclair and Lo 19861 and since most of the cortical input to the HF arrives via the dentate gyrus (DG) (Anderson et ai. 1966; Lomo 1971; Hjorth-Simonsen and Jeune 1972; Amaral and Witter 19891, thereby affording an opportunity for modification of cortical signals before they diverge into numerous limbic circuits (Cotman 19781, the DG in the anterior HF was chosen as a strategic infusion site to influence pain processing. The formalin test was used to quantify pain since it provides a valid experimental model of injury-induced pain in humans (Melzack 1989).

Methods Subjects and housing Male Long-Evans rats weighing 330-390 g at the time of surgery were used as subjects in this experiment. Animals were housed

106 individually in a colony room maintained on a 12-h light schedule (lights on at 07.00 h). with food and water available ad libitum in home cages.

Surgery and testing procedures Rats were anesthetized with sodium pentobarbital(60-70 mg/kg) and administered atropine sulphate (0.4 mg/kg) to reduce mucous secretions. An 1 l.O-mm guide cannula constructed from a 23-gauge (0.7 mm) needle was unilaterally implanted into the cortex dorsal to the DG. Stereotaxic coordinates employed were: 2.5 mm posterior to bregma, 0.6 mm lateral from the midline, and 2.0 mm ventral to the lambda-bregma plane (Paxinos and Watson 1986). The cannula was fastened to the skull by 3 jewellers’ screws and a headstage made of dental acrylic. A size 00 stainless steel stylet (0.3 mm), lowered into the guide cannula until it extended 2.5 mm ventral to the tip. was fastened with a drop of cement.

Test apparatus and procedure The observation box used in the formalin test consisted of a two-chambered plexiglass box. The upper chamber measured 32 x 32 X 32 cm with a plexiglass floor perforated with &mm holes spaced 3X mm apart. The lower chamber held a large mirror positioned diagonally. so that an unobstructed view of the animal’s paws was available to the experimenter seated in front of the box. The animals were habituated to the test box for I h on each of the 3 days prior to testing. On the day of testing, each animal was again placed within the observation box to habituate for 20-30 min prior to receiving a SO PI subcutaneous injection of 2.5% buffered formalin acetate into the plantar surface of the rear paw contralateral (n = 28) or ipsilateral (n = 28) to the cannula for infusion into the DG. The animal was then returned to the observation box for 20 min prior to receiving a single intracranial microinjection.

Drugs and microinjection procedure Each animal was hand-held while the stylet was withdrawn and replaced with a 13.6 X 0.4 mm injection cannula (30 gauge stainless steel tubing) which extended 2.6 mm ventral to the tip of the guide cannula. The inner cannula was connected via 30 cm of PE-IO polyvinyl tubing to a 5.0-~1 Hamilton syringe, so that the animal could move about freely during drug administration. The rat was placed inside an open 20 x 24 x 45 cm box, where it received a single 1.0~~1 injection of either 2% lidocaine or 0.9% sterile saline (without preservatives) slowly over 2 min, after which the injection cannula was left in place for an additional I-min period to prevent reflux. The injection cannula was then withdrawn, the stylet was replaced inside the guide cannula and the animal was returned to the observation box prior to behavioral scoring.

The formalin test The formalin test has been described in detail elsewhere (Dubuisson and Dennis 1977). Briefly, the formalin injection causes a biphasic pain reaction consisting of an initial sharply rising, fairly intense pain, which diminishes after approximately 5-10 min and is replaced by an apparently more moderate pain. This second phase rises to a stable level after approximately 20 min and slowly wanes over the subsequent 60-90 min. Behavioral observations in this experiment commenced 30 min after formalin injection - during the second, more stable pain phase - and ended 40 min later (i.e.. 70 min after formalin injection). A modified 4-point rating scale, previously described by Cohen et al. (1984) was used to quantify the animal’s behavior. In summary. a behavioral score of 0 denotes normal use of the injected paw (i.e.. the plantar surface is flat on the floor of the observation box, and the animal’s weight is evenly distributed between hind paws). A score of

I indicates favoring of the injured paw. with some part of the paw in contact with the floor. A score of 2 indicates elevation of the paw. A score of 3 denotes licking or chewing of the injured paw, which is distinct from normal grooming behavior. Behavioral scores were continuously entered into a Radio Shack TRS-X0 minicomputer for the JO min of the test. A weighted average pain score for each 5-min interval was calculated by multiplying the time spent in each category by the category weight and then dividing by the total time in that period.

Control experiments To control for the possible absorption of the local anesthetic into adjacent neural structures, as well as the possibility that the intracranial microinjection procedure itself might cause a change in behavior. formalin pain scores were evaluated for animals whose injection cannulae were located in structures adjacent to the DG. In order to investigate the temporal factor of lidocaine injection as it relates to pain scores in the formalin test, an additional group of animals was administered a I.O-~1 intracranial microinjection of 2% lidocaine or 0.75% bupivacaine (a longer-acting local anesthetic) IO min before administration of the formalin injection (i.e., 40 min prior to the commencement of testing). All other procedures were identical to those previously described. Following testing. each animal was administered an overdose of chloral hydrate and perfused with 0.9% saline followed by IO% formalin. The guide cannula and headstage were cut away from the skull and the animal’s brain was removed and stored in formalin for at least 24 h. Any animals whose brains showed excessive damage along the cannula track were excluded from statistical analysis. C‘annula placements were verified from 30-wrn coronal sections stained with thionin. Only if the inner cannula tip was clearly located in the DG or in an adjacent structure was that animal included in the DG or appropriate control group.

Statistical analysis Data obtained in the formalin test were examined for differences in average pain scores among groups across 5. 20. and 40-min time intervals using the Kruskal-Wallis H statistic. Mann-Whitney U tests were employed to evaluate differences between pairs of treatment groups at various time intervals.

Results Fig. 1 indicates the location of cannula tips for those animals (n = 29) that received a microinjection in the DG following formalin injury. Cannula tips were located 1.8-3.1 mm posterior to bregma. Of these animals, 9 received lidocaine contralateral and 9 received lidocaine ipsilateral to the injured paw. In the control condition, 8 animals received contralateral and 3 animals received ipsilateral saline injections into the DG. A preliminary analysis of the data revealed no difference due to laterality of saline injection, and thus a combined saline control group (n = 11) was used for subsequent comparisons. Fig. 2 shows formalin test scores for contralateral and ipsilateral drug groups compared to the saline control group. No difference was evident among the 3 groups during the first 20 min of the test period. However, the pain scores for the 3 groups diverged during the last 20 min of the test (H = 7.7. P = 0.02),

107

-3.14mm

- 2.30 mm

-2.12 mm

Bregma - l.80 mm

Fig. 1. Location of inner cannula tips is depicted for each of 29 rats that received a microinjection in the dentate gyrus 20 min after formalin injury. Drawings are from the Paxinos and Watson atlas (1986). Symbols on the left side of the atlas plates represent contralateral treatments and on the right ipsifateral treatments. Cannula tips were located in the center of the symbols shown: 0, lidocaine; 0, saline.

10X

with the contralateral lidocaine group exhibiting a substantial decrease in pain scores relative to the saline group during 50-55 min (Z = 2.20, P < 0.03), 55-60 min (Z = 2.40, P < 0.02), 60-65 min (Z = 2.91, P < 0.01) and 65-70 min (Z = 2.10, P < 0.04) time intervals. The ipsilateral lidocaine treatment group had a significantly lower pain score than the saline group only during the 65-70 min interval (Z = 2.67, P < 0.01). Lidocaine infusions into the DG caused no discernable differences in motor coordination or activity level compared to normal animals.

the DG pre-injury anesthetic (Z = 3.18, P < 0.01) adjacent pre-injury anesthetic (Z = 2.23, P < 0.03) adjacent post-injury anesthetic (Z = 2.54, P < 0.02) and saline (Z = 2.12, P < 0.05) treatment conditions). The ipsilateral DG post-injury lidocaine treatment - which showed a similar (although non-significant) trend toward lower pain scores during the last half of the formalin test - was not significantly different from the contralateral lidocaine post-injury treatment (Z = 1.89, P < 0.06).

Control experiments for site and time of injection Fig. 3 indicates the location of the cannula tips in the control experiments. Tips were located in the DG (n = 6), or in adjacent structures (n = 21), including the habenula, stria medullaris, dorsal hippocampus, hippocampal commisure, dorsal superior colliculus and mediodorsal thalamus. Mann-Whitney U Tests indicated that none of the other control treatments (i.e., groups that received infusions of lidocaine in structures adjacent to the DG (n = 11) after formalin injury or groups that received bupivacaine (n = 7) lidocaine (n = 5) or saline (n = 4) infusions prior to injury) resulted in lower pain scores than the saline control group during the O-20 or 20-40 min test intervals. Fig. 4 illustrates the relative efficacy of each treatment condition during the 20-40 min test interval. A Kruskal-Wallis test indicated non-homogeneity among the average pain scores of all treatment groups during the 20-40 min interval (H = 13.7, P < 0.02) and among overall pain scores (H = 17.3, P < 0.01). U tests revealed that the contralateral lidocaine post-injury treatment group had a significantly lower overall pain score than each of the control treatment groups (i.e.,

Discussion

Pain Scores Following Dentate Gyms Infusion

3

-

:

30-35

35-40

40-45

Salme lpsl +contra (n-11)

-

Lidocame

ipsilateral

-D-

Lidocaine

contralateral

45-50

Time After Formalin

50-55

55-60

(n=9)

60-65

(n=9)

65-70

Injection (minutes)

Fig. 2. Average pain scores during 5-min intervals for animals administered a 1.0.~1 injection of lidocaine or saline into the dentate gyrus 20 min after subcutaneous formalin injection. Significant differences from saline control group are denoted by: * P < 0.05, Mann-Whitney U).

The results indicate that regional anesthesia of the dentate gyrus (DG) of the hippocampal formation (HF) produces an analgesic effect in the formalin test. The HF is, therefore, implicated as a neural structure involved in tonic pain perception. The analgesia which results from this treatment is not due to a neurotoxic effect of the lidocaine (Kalichman et al. 1988) since there was no behavioral difference between the group which received microinfusion of the anesthetic prior to formalin injury and the saline control group. It has also previously been shown that when lidocaine is centrally administered 60 min before subcutaneous formalin injection, there is no difference between drug and saline treatment conditions (Vaccarino and Melzack 1989a,b). These results indicate that lidocaine’s analgesic effects are due to the temporary interruption of central neural activity rather than from permanent destruction of neurons. The analgesia produced by anesthetization of the hippocampal DG is not likely to be due to diffusion of lidocaine into structures outside the HF. It has previously been reported that the functional spread of lidoCaine can range from less than 1 mm in the oculomotor nucleus for a 4-~1 infusion (Albert and Madryga 1981) to almost 3 mm for a I+1 infusion in the dorsal columns of the spinal cord (Sandkuhler et al. 1987) (discrepencies may be due to differences in the volume or rate of anesthetic infusion, physiological differences of neurons at diverse sites within the central nervous system (CNS) or differences in sensitivity of the testing paradigms used). Within the present experiment, data from the 21 animals that received local anesthetic infusions in structures surrounding the DG was used as a gauge of the functional spread of the drug. The data indicate that the analgesic effect of lidoCaine infusions in the DG were probably not due to diffusion of the drug to structures adjacent to the HF, since direct infusion at these sites caused no discernable changes in pain behavior; nor is it likely that this effect was due to absorption of the drug at a more distal site of action, since diffusion from at least some of the aforementioned 21 infusions must be assumed

109

-630mm

- 6.04 mm

- 3.30 mm

-3.14mm

- 2.30 mm

- 1.80 mm

Bregmr - 1.40 mm

Fig. 3. Location of the inner cannula tips is depicted for each of the 27 rats in the control experiments. Drawings are from the Paxinos and Watson atlas (1986). Symbols on the left side of the atlas plates represent contralateral treatments, while those on the right represent ipsiiateral treatments. Cannula tips were located in the center of the symbols shown: 0, saline; 6, lidocaine 20 min after injury; 0, lidocaine 10 min pre-injury; n , bupivacaine 10 min pre-injury.

Pain Scores SO-70 Min After Formalin Injury q El

Lidcraine in rontra DG Lidtraine in ipsi DG

0 H

Saline in DC Lidtraine Adjacent to DG

n

Lido or Bupiv in DC

Post-injury Fig. 4. Average formalin

pain

scores during

test for animals administered

or saline into the dentate prior or 20 min subsequent ence from

the saline

Pre-injury the last 20-min

gyrus or into adjacent to formalin

treatment

structures.

injury. A significant

is denoted

Whitney

period

of the

a 1.0~@I injection of lidocaine

by: * P < 0.05,

IO min differMann-

U).

equally capable of reaching such a site. Finally, none of the pre-injury anesthetic infusions within the DG reduced pain behaviors, despite the fact that diffusion factors in this condition would be identical to the post-injury infusions which were effective in reducing pain behavior. The present findings are in agreement with earlier experimental data which indicate that lidoCaine infusion into the lateral hypothalamus produces analgesia in the formalin test, while infusion into the adjacent medial hypothalamus does not (Tasker et al. 1987). Similarly, lidocaine produces analgesia when injected into the cingulum bundle but not into sites in the adjacent cingulate cortex (Vaccarino and Meizack 1989a). Clearly, these effects are localized to a specific set of structures. A number of studies have indicated that a microinjection of lidocaine in the CNS attains its maximum effect within lo-15 min of administration and wanes in approximately 40-50 min (Albert and Richmond 1976; Albert and Madryga 1981; Sandkuhler and Gebhart 1984; Sandkuhler et al. 1987). The analgesia which resulted from injection of the cingulum bundle occurred within this interval (Vaccarino and Melzack 1989a,b). Another study, however, found that lidocaine injected into the lateral hypothalamus did not cause significant analgesia in the formalin test until 30 min after infusion (Tasker et al. 1987). The delay in analgesia evident in the latter study as well as that seen in the present study should not be interpreted as meaning that lidocaine did not block neural transmission until 30 or 40 min after microinjection, but rather that the behavioral consequences of the blockade of neural processing in these structures were not manifested in the formalin test until that time.

This situation is analogous to experimental data which demonstrate that stimulation of an area of the CNS can cause changes in neuronal function and subsequent behavior (e.g., the memory-improving effects of post-training amphetamine injections in the HF arc evident long after the injected substance has been metabolized (Packard and White 1989). Similarly, the reduction in autotomy over a 70-day period which resulted from 5 infusions of 1.0 ~1 of bupivacaine into the cingulum bundle during a 21-day period following peripheral neurectomy demonstrates that anesthetic blockade of neural activity may have consequences which far outlast the pharmacological activity of the anesthetic (Vaccarino and Melzack 1991). If pain information undergoes prolonged processing through a series of neural networks over a period of time, then the deactivation (or partial deactivation) of a circuit within that network - which presumably adds to (or maintains) the overall intensity of the complex pain signal may result in a gradual decrease in the strength of that signal and thus produce an incremental analgesia. There is recent evidence that pain behavior cxpressed in the late phase of the formalin test is not solely dependent upon peripheral input but may also rely to a large extent on plastic changes within the CNS induced by nociceptor-generated activity which has occurred during the earlier phase (Coderre et al. 1990). The DG may be a component within a neural network which is triggered only when nociception persists bcyond a certain minimum duration. Such a mechanism might be hypothesized as necessary in order to activate the adaptive affective and behavioral responses to tonic pain which are not manifested during phasic pain (Melzack and Wall 1988; Melzack 1990). Because these responses are presumed to involve limbic forebrain structures, and since one of the putative mnemonic functions of the HF involves the incorporation of temporal data into memory traces (Jacobs and Nadel 1985), one could speculate that the DG serves to collate the critical temporal information with information from other neural networks that process pain-related and other information. Data from the pre-injury treatment condition may be interpreted as evidence that the functional block of DC neural circuitry can cause significant analgesia during the formalin test only if administered during a particular time interval. The ineffectiveness of local anesthetic infusion in the DG prior to formalin injury in producing subsequent analgesia may indicate that the HF is not critically involved in the perception of the initial phasic pain arising from the formalin injection. This could be interpreted as further evidence that phasic and tonic pain are each subserved by discrete neural substrates (Dennis and Melzack 1977). The difference in analgesic potency of lidocaine infusions in the DG as a function of the laterality of

111

injury is an interesting result in the context of previous studies involving central anesthetic-induced analgesia. Contralateral treatment caused significant analgesia in this experiment, while ipsilateral treatment resulted in a trend toward lower pain scores which did not achieve statistical significance. Similarly, a stronger contralatera1 than ipsilateral analgesia resulted from lidocaine block of the cingulum bundle (Vaccarino and Melzack 1989a), while equilateral analgesia resulted from infusion of lidocaine into the lateral hypothalamus (Tasker et al. 1987). These inter-experimental differences in analgesic potency as a function of the laterality of anesthetic infusion may be due to variations in the degree of somatotopic organization of afferent signals received by each of the target structures or in the completeness of the neural blockade relative to the overall size of the structure. Finally, since regional anesthetic block affects both neurons and fibres of passage, the present study does not rule out the possibility that deactivation of the latter is the basis for the reduction in pain behavior observed subsequent to the administration of lidocaine in the DG. This possibility does not, however, diminish the authors’ assertion that the HF processes information related to tonic pain perception, since virtually all of the fibres traversing the DG region are believed to be HF afferents or efferents (Anderson et al. 1966; L@mo 1971; Hjorth-Simonsen and Jeune 1972; Swanson and Cowan 1977; Swanson et al. 1978; Amaral and Witter 1989). Although further experimentation is required in order to explain these experimental results, it is evident from the data that anesthetization of a region of the HF is capable of reducing a pre-existing tonic pain. This analgesic effect is incremental in its onset and is considerably stronger when administered to the side contralateral to the site of injury.

Acknowledgement

This work was supported by NSERC Grant A7891.

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