Brainstem Regions Affecting Minimum Alveolar Concentration and Movement Pattern During Isoflurane Anesthesia

Brainstem Regions Affecting Minimum Alveolar Concentration and Movement Pattern during Isoflurane Anesthesia

Steven L. Jinks, Ph.D.,* Milo Bravo, B.S.,† Omar Satter, M.B.B.S.,‡ Yuet-Ming Chan, B.S.§ Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021

ABSTRACT Background: Spinal transection or selective delivery of volatile an- What We Already Know about This Topic esthetics to the spinal cord reduces minimum alveolar concentration ❖ (MAC), whereas precollicular decerebration does not. The authors Minimum alveolar concentration (MAC) is defined by lack of movement to incision and largely reflects anesthetic inhibition sought to determine which brainstem regions influence anesthetic in the spinal cord requirements and movement responses with isoflurane. ❖ Brainstem–spinal circuits facilitate movement to incision, but Methods: Movement (biceps femoris electromyogram) and MAC the sites relevant to MAC are not well known were measured in adult rats before and after decerebration at the precollicular, mid-collicular, pontine or medullary level, or decer- What This Article Tells Us That Is New ebellation. Additional experiments assessed the effects of lidocaine inactivation of the mesencephalic locomotor region on MAC ❖ In normal rats, MAC is reduced by injury or inhibition of the and the effects of isoflurane on nociceptive neuronal responses in mesencephalic locomotor region, suggesting that this motor this region. regulatory site counteracts anesthetic actions in the spinal Results: Transections placed at the level of the mid-colliculus, ros- cord for immobilization to surgery tral pons, and pontomedullary junction significantly reduced MAC by approximately 10, 40, and 45%, respectively. MAC was decreased 9% after mid-medullary transections that were placed caudal to the OLATILE anesthetics act primarily in the spinal cord to nucleus raphe magnus but rostral to the dorsal reticular nucleus; Vabolish movement in response to noxious stimula- however, only weak, single movements occurred. Caudal medullary tion.1–3 However, selective delivery of isoflurane to the spinal transections at the obex decreased MAC by 60%. Bilateral inactivation of the mesencephalic locomotor region with lidocaine caused a cord (keeping cranial isoflurane concentration low) reduces reversible, 32% decrease in MAC and reduced the number and isoflurane minimum alveolar concentration (MAC) by more amplitude of movements at sub-MAC isoflurane concentrations. than 30%.4 Moreover, in rats, chronic spinal transection Neuronal responses of mesencephalic locomotor region neurons to reduces MAC by approximately 50% in the absence of spinal supramaximal noxious tail clamp were reduced by 87% by 1.2 MAC shock.3 This means that the supraspinal regions contribute isoflurane. Conclusions: The authors conclude that the mesencephalic loco- to determining anesthetic immobilizing requirements, ap- motor region influences anesthetic requirements and promotes re- parently by counteracting a direct depressant action in the petitive movement with sub-MAC isoflurane by facilitating ventral spinal cord through descending facilitation. Because precol- spinal locomotor circuits, where anesthetics seem to exert their key licular decerebration does not significantly change MAC or immobilizing effects. However, net brainstem influences on MAC the type of movement elicited by a noxious stimulus at sub- seem to result from interaction among descending nociceptive and 5,6 locomotor modulatory pathways. MAC isoflurane concentrations, the important supraspinal sites responsible for determining anesthetic requirements in an intact animal lie in the brainstem. * Assistant Professor, † Staff Research Associate, ‡ Junior Special- ist, § Undergraduate Assistant, Department of Anesthesiology and One candidate site is the mesencephalic locomotor re- Pain Medicine, University of California School of Medicine, Davis, gion (MLR), based on its ability to initiate locomotion California. through descending facilitation of locomotor circuits in Received from Department of Anesthesiology and Pain Medicine, the ventral horn,7 where anesthetics predominantly act to University of California, Davis, Davis, California. Submitted for pub- 6,8–10 lication June 1, 2009. Accepted for publication October 7, 2009. produce immobility. Furthermore, it has been Supported by R01 GM 078167 and R01 GM 061283 from the National shown that noxious stimulation sufficient to elicit motor Institutes of Health, Bethesda, Maryland, and the University of reflexes evokes neuronal responses in mesencephalic areas California, Davis Department of Anesthesiology and Pain Medicine. associated with the MLR,11 namely the cuneiform and Address correspondence to Dr. Jinks: TB-170, Department of Anes- 12 thesiology and Pain Medicine, University of California School of Med- pedunculopontine nuclei. icine, Davis, California 95616. [email protected]. Information on pur- We hypothesized that MAC values would decrease on chasing reprints may be found at www.anesthesiology.org or on the removal of descending locomotor facilitation: after brain- masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 stem transections associated with removal of the MLR or months from the cover date of the issue. during local inactivation of the MLR with lidocaine micro-

Anesthesiology, V 112 • No 2 316 February 2010 PERIOPERATIVE MEDICINE 317 injection. We also tested whether peri-MAC concentrations Caudal medullary transections were based on viewing the reduced this descending facilitation by examining the effect obex. Brain structures rostral to the transection were aspi- of isoflurane on noxious stimulus-evoked responses of MLR rated, and gelfoam was gently placed against the cut end of neurons. We further hypothesized that MAC in animals with the transection. We performed only one transection in each more caudal, mid-medullary transections would be greater animal. than in animals with pontine/rostral medullary transections. Biceps femoris electromyogram signals were amplified This is based on removal of the rostral ventromedial medulla and band-pass filtered (10 Hz to 2 kHz) with a Tektronix (RVM), where peri-MAC isoflurane produces a net increase differential amplifier (model 2601; Beaverton, OR). Electro- in descending inhibition.13 Finally, caudal medullary tran- myogram was fed to a Cambridge Electronic Design, Power sections that remove the dorsal reticular nucleus (MdD), a 1401 data acquisition system with Spike 2 software (Cam- 14,15 pronociceptive area, should then decrease MAC to val- bridge Electronic Design, Cambridge, United Kingdom). Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021 ues seen in spinally transected animals.3 MAC Determination Materials and Methods We recorded electromyogram responses and determined MAC in each animal before and more than 90 min after a The University of California, Davis Animal Care and Use decerebration at the level of the mid-colliculus, rostral pons, Committee (Davis, California) approved this study. Animals pontomedullary junction, mid-medulla, and caudal medulla were given free access to food and water and maintained on a or after decerebellation. MAC was determined using a tail 12/12-h light–dark cycle with lights on at 7:00 AM. All ex- clamp. Animals were anesthetized with isoflurane, and the periments were conducted on adult male Sprague-Dawley clamp was applied for up to 1 min or until movement was rats (400–550 g). observed. A positive response was based on observation of multisegmentally mediated movement (movement of a Surgery and Setup limb or limbs in response to tail clamp). Typically, head Anesthesia was induced in an acrylic box with isoflurane turning is also considered a positive response in MAC (5%), followed by intubation with a 10-gauge catheter and testing, which was not assessed in the current study be- mechanical ventilation with isoflurane mixed in 100% oxy- cause the experiments necessitated fixing the head to the gen. Body temperature was monitored and maintained at stereotaxic frame. However, in our experience, head turn- 37° Ϯ 1°C with a heating pad. End-tidal carbon dioxide and ing in the absence of limb movement is less common, and anesthetic concentration were monitored continuously with our MAC testing criteria yielded typical baseline MAC an Ohmeda Rascal II analyzer (Helsinki, Finland). values (see Results). Depending on the response, the an- A carotid artery and a jugular vein were each cannulated esthetic concentration was changed in 0.2% increments to permit blood pressure recording (model PB-240; Puritan- with an intervening 15–20 min equilibration period. The Bennett Corp., Hazelwood, MO) and fluid administration, average of the two values that just permitted and just respectively. Mean arterial pressure was maintained at more prevented movement was MAC. than 60 mmHg using lactated Ringer’s solution and/or hetastarch when necessary. Dexamethasone (1 mg/kg, intra- MLR Identification and Lidocaine Microinjection venously) was administered at the beginning of surgery to In precollicular decerebrate animals, an Ag-AgCl stimu- minimize brain swelling and trauma. Platinum needle elec- lating electrode was placed inside a glass pipette (outer tip trodes were inserted bilaterally into the biceps femoris mus- diameter: 50–100 ␮m) filled with saline alone or 4% lido- cles and sutured in place to record electromyogram activity. caine (Sigma, St. Lois, MO) in saline. The injection pipette/ The animal was fixed in a stereotaxic frame with an incisor electrode was lowered into the midbrain to search for the clamp, earbars, and the body supported with a sling. During MLR using electrical stimulation. By using the center of the deep isoflurane anesthesia, a craniotomy was made between intercollicular crux as a zero reference point, we positioned bregma and lamda, and a decerebration was made at different the stimulating electrode 1.8–2.0 mm lateral to the midline levels ranging from the rostral end of the superior colliculus and Ϯ0.5 mm anterior-posterior from this point. Constant- to the trigeminal nucleus caudalis (at the obex). In one group current electrical pulses (0.5 ms pulse duration, 60 Hz) were of animals, we aspirated the entire cerebellum while leaving passed through the electrode using a PSIU6 stimulus isola- the remainder of the brain intact. tion unit connected to an S88 stimulator (Grass-Telefactor, Decerebrations were performed by making a large crani- Warwick, RI). When a site was found to elicit locomotion, otomy between bregma and lambda, aspirating the cortex to we decreased and increased the stimulus intensity and finely view subcortical structures, and transectioning the brain with adjusted the position of the electrode to determine the lowest a scalpel blade. Precollicular, mid-collicular, and rostral pon- threshold site for four-limb galloping (threshold range: tine (immediately post collicular) transections were based on 20–60␮A). viewing the colliculi. Pontomedullary and mid-medullary After MLR identification on each side of the midbrain, transections were referenced to bregma at coordinates of lidocaine (4%, 1.0 ␮l) was injected into the MLR bilaterally Ϫ10 to Ϫ11 mm and Ϫ13.0 to Ϫ14.0 mm, respectively. at 0.8 MAC, and tail clamp was applied every 5–10 min until

Jinks et al. Anesthesiology, V 112 • No 2 • February 2010 318 Brainstem Influences on MAC and Movement Pattern recovery was observed. If the animal failed to respond to the injection group and for the mid-medullary transection group tail clamp (negative MAC test), after recovery, the isoflurane using a two-tailed paired t test. Comparisons between groups concentration was decreased by 0.2 MAC, and the lidocaine of animals were made using a two-tailed unpaired t test. A injections were repeated. In pilot studies, a 0.5-␮l volume of one-way ANOVA with “neuron” as a random effects factor, lidocaine (4%) decreased MAC in 50% of animals only, followed by Tukey multicomparisons, was used to compare whereas a 1.0-␮l volume decreased MAC to more than 20% changes in MLR neuronal activity across isoflurane concen- in all animals tested. trations. A P value less than 0.05 was considered statistically significant. Data analyses were performed using SPSS (Chi- Electrophysiology cago, IL). During isoflurane anesthesia (1.5–1.8%), we performed a precollicular decerebration and a lumbar laminectomy to Results Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021 dissect free L4–5 ventral roots. After a 90-min postdecer- ebration recovery period and verification that MAC had re- Effects of Brainstem Transection and MLR Inactivation turned to more than 90% of control, we searched for the on Isoflurane MAC MLR using low-threshold electrical stimulation as previously Pretransection (intact) isoflurane MAC values were 1.3% described.6 We then paralyzed the animal with pancuronium atm Ϯ0.1% SD. Transections placed at the mid-collicular Ϫ Ϫ bromide (0.6 mg ⅐ kg 1 ⅐ h 1), cut ventral roots distally, and level (n ϭ 9) caused a small but significant decrease in MAC placed them bilaterally on platinum hook electrodes that to 90 Ϯ 5% SD of intact MAC values (P Ͻ 0.006). Tran- were insulated with a vaseline and mineral oil mixture for sections placed in the pons, immediately caudal to the infe- electroneurogram recording. Ventral root electroneurogram rior colliculus (n ϭ 8) or near the pontomedullary junction activity was recorded to monitor motor output while record- (n ϭ 9), substantially decreased MAC to 60 Ϯ 9% SD and ing single-unit activity in the MLR. The tungsten MLR mi- 55 Ϯ 5% SD of intact MAC values, respectively (P Ͻ 0.0006 croelectrode (8–10 M⍀, FHC, Bowdoinham, ME) was in both cases). However, when transections were placed cau- switched from stimulation to recording, and we isolated a dal to the nucleus raphe magnus/gigantocellularis pars alpha, single neuron that responded to noxious mechanical tail but rostral to the MdD (n ϭ 15), MAC decreased to only stimulation. Single-unit activity was band-pass filtered (300 91 Ϯ 8% SD of intact MAC values (P Ͻ 0.03). Isoflurane Hz to 5 kHz) with a Grass p511 amplifier (Grass-Telefactor, MAC dramatically decreased to 40 Ϯ 3% SD (P Ͻ 0.0006) Warwick, RI) and acquired on a PC using a Power 1401 in animals that received obex-level transections, at the caudal system with Spike 2 software. After a MLR unit was isolated, edge of the MdD and within the trigeminal nucleus (n ϭ 6). supramaximal noxious mechanical stimulation (tail clamp) Animals that received a complete cerebellectomy did not was applied for 30 s with 0.0, 0.4, 0.8, and 1.2 MAC isoflu- exhibit a significant change in their MAC values (95 Ϯ 9% rane. The total number of action potentials and peak firing SD of intact MAC; n ϭ 7). Figure 1 shows individual exam- rate during tail stimulation were measured at each isoflurane ples of movement/MAC changes after a rostral transection of concentration. the pons (fig. 1A) and after a mid-medullary transection (fig. 1B). Figure 2A shows mean MAC values in separate groups Histology of animals receiving different levels of brainstem transec- At the end of experiments, animals were killed with satu- tions, lidocaine microinjection into the MLR, or cerebellec- rated KCl with isoflurane, and the remaining brainstem was tomy. Figure 2B shows the range of transection levels of each removed and placed in formalin for at least 24 h, followed by group that we histologically examined. 30% sucrose. Brainstems were cut sagittally, and sections Bilateral inactivation of the MLR by lidocaine microin- from several mediolateral levels on each side were mounted, jection (n ϭ 10) caused a 32 Ϯ 6% decrease in MAC (P Ͻ counterstained with cresyl violet, and coverslipped. These 0.0001) that recovered 20–60 min after injection. The line sections were used to verify the level of transection. If the graph in figure 2 shows mean MAC values after lidocaine intended transection was actually placed at the level of an- microinjection into the MLR. An individual example show- other group, that animal was reassigned to the appropriate ing the reversible effect of MLR lidocaine microinjection on transection group. In three cases, transections were found to tail clamp-evoked hindlimb electromyogram activity is be at a level not consistent with any of the transection groups, shown in figure 3. As a control for lidocaine spread, six ani- and these animals were excluded from the study. mals received 0.5 ␮l lidocaine (4%) injections 700 ␮m dorsal and 700 ␮m ventral to the site producing locomotion in Statistics response to low-threshold electrical stimulation, and in no Changes in MAC for each transection group were assessed cases did these injections change MAC (n ϭ 6). by comparing posttransection MAC with each group’s re- spective intact MAC value using a two-tailed paired t test Effects of Brainstem Transection and MLR Inactivation with a Bonferroni correction for multiple comparisons. on Noxious Stimulus-evoked Movement Pattern Within-group comparisons of movement number and peak Under 0.6 MAC (pre-lidocaine injection MAC), micro- movement amplitudes were made for the MLR lidocaine injection of lidocaine into the MLR significantly reduced the

Anesthesiology, V 112 • No 2 • February 2010 Jinks et al. PERIOPERATIVE MEDICINE 319

AB0.8 MAC 0.6 MAC Clamp Tail Pre-transect Clamp Tail Post-transect

R. BF R. BF

5 sec 5 sec L. BF L. BF

C 0.8 MAC D 0.8 MAC Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021 Clamp Tail Pre-transect Clamp Tail Post-transect

R. BF R. BF

5 sec 5 sec L. BF L. BF

Fig. 1. Pontine transections decrease minimum alveolar concentration (MAC). Transections made in the pons or pontomedullary junction block multisegmental movement at sub-MAC isoflurane concentrations. (A) Baseline biceps femoris electromyogram motor response to a 30-s tail clamp in an intact animal. R.BF ϭ right biceps femoris; L.BF ϭ left biceps femoris. (B) Negative response in the same animal with pontine transection (indicated by dashed line in diagram). Mid-medullary transection minimally decreases MAC but prevents repetitive movement during tail clamp. (C) Repetitive movement elicited by a 30-s noxious tail clamp in another intact animal (D) Response to tail clamp in the same animal after transection of the mid-medulla at the rostral end of the medullary dorsal reticular nucleus (dashed line in upper right). The transection did not change isoflurane MAC in this animal, but only single low-magnitude movements occurred at the onset and offset of the noxious tail clamp. amplitude (P Ͻ 0.012) and number of movements (P Ͻ MAC isoflurane was significantly reduced to 16 Ϯ 15% SD 0.02) elicited by a supramaximal noxious mechanical tail of 0.8 MAC values, and peak firing rate was reduced to 49 Ϯ stimulus (fig. 4). No animals displayed positive movement at 22% SD (fig. 5). From 0.0 to 0.8 MAC, the total number of 0.8 MAC after lidocaine injection. Low levels of tonic elec- spikes was not changed significantly; however, peak firing tromyogram activity were often detectable, but this did not rate was significantly decreased to 74 Ϯ 8% SD of control translate to observable movement (criterion for a positive (P Ͻ 0.034). An individual example of isoflurane effects on response in MAC testing). Effects of MLR lidocaine micro- tail clamp-evoked responses of an MLR neuron is shown in injection on mean amplitude and number of movements are figure 5A. Mean isoflurane effects on MLR neurons (n ϭ 11) shown in figure 4. are shown in figure 5B, and histologically identified record- Although MAC was only modestly reduced after mid- ing sites are illustrated in figure 5C. medullary transections, movement was weaker and nonrepetitive, where animals only displayed a single movement at the onset and/or offset of tail clamp application. Discussion Both movement number and amplitude with 0.8 MAC isoflurane were significantly reduced in this group (n ϭ 6) We investigated the influences that different brainstem compared with intact control values (P Ͻ 0.03 in both regions had on isoflurane MAC and noxious stimulus- cases). evoked movement patterns and isoflurane effects on movement-related neurons in the MLR. Although volatile Effects of Isoflurane on Neuronal Activity in the MLR anesthetic-induced immobility is explained by a direct Neuronal responses to supramaximal noxious mechanical anesthetic action in the spinal cord, the literature collec- tail stimulation exhibited bursting behavior that was associ- tively suggests that the brainstem, but not forebrain re- ated with movement bouts detected in ventral root record- gions, indeed plays a critical role in establishing the pre- ings under sub-MAC isoflurane concentrations. Isoflurane cise anesthetic immobilizing requirements in an intact significantly reduced the total action potentials and peak animal. This was shown in previous studies reporting that firing rate of MLR neuronal responses (n ϭ 11) to tail clamp precollicular decerebration does not change MAC,5,6 at 1.2 MAC, compared with responses recorded under anes- whereas chronic spinal transection, reversible spinal cold thetic-free baseline (0.0 MAC), 0.4 MAC, and 0.8 MAC block, and selective perfusion of the spinal cord (in goats (P Ͻ 0.001 for all comparisons). With 1.2 MAC isoflurane, with an intact nervous system) all decrease MAC by 30– MLR total action potentials were reduced to 13 Ϯ 9% SD of 50%.3,4 Overall, our data suggest that facilitation of spinal control (0.0 MAC), and peak firing rate was reduced to 34 Ϯ locomotor networks from the MLR, nociceptive inhibi- 14% SD. The total number of action potentials with 1.2 tion from the RVM, and nociceptive facilitation from the

Jinks et al. Anesthesiology, V 112 • No 2 • February 2010 320 Brainstem Influences on MAC and Movement Pattern

A A Elect. Stim, Inject Lido MLR Stim 60 µA 100 + % 90 R. BF

80 MLRMLR 5 sec 70 * ** L. BF 60 50 B Clamp Tail Pre-lidocaine $

40 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021

CNCN R. BF % INTACT MAC % INTACT 30 0.8 PPNPPN MAC 5 sec 20 L. BF Vc

10 7 Clamp Tail 6 min Post-lidocaine 0 C Pre- Mid--MLRMLR Ponto Mid Obex R. BF coll coll lido med Med 0.8 MAC B L. BF D Clamp Tail 60 min Post-lidocaine 0.8 R. BF CN MAC L. BF 1 PPN

4 Fig. 3. Midbrain locomotor region (MLR) inactivation reduces mini- 2 3 mum alveolar concentration (MAC). (A) Electrical stimulation was used to perform a functional search for the MLR, and lidocaine was injected bilaterally at sites producing the low-threshold hindlimb lo- Fig. 2. Isoflurane minimum alveolar concentration (MAC) after differ- comotion (right). (B) Normal electromyogram hindlimb motor re- ent lesions of the brainstem and inactivation of the midbrain locomo- sponse to noxious tail clamp. (C) The same animal was immobile tor region (MLR) by lidocaine. Data are shown as mean and SEM (A) during tail clamp with 0.8 MAC isoflurane after bilateral lidocaine Mean isoflurane MAC values. Dashed lines denote transections. Pre- microinjection into the MLR. (D) Robust and repetitive tail clamp- collicular decerebration did not significantly change MAC. MAC was elicited movement recovered 60 min after MLR lidocaine microinjec- slightly but significantly reduced to 90% of control (ϩ, P Ͻ 0.006) tion. R.BF ϭ right biceps femoris; L.BF ϭ left biceps femoris. after mid-collicular transections that began to encroach on or removed part of the pedunculopontine nucleus (PPN) associated with the MLR. Inactivation of the MLR with bilateral lidocaine microinjec- of isoflurane on these regions, and the limitations of the tions (shaded) decreased MAC to 68% of control (* P Ͻ 0.001), study. whereas transections ranging from the rostral pons to the pon- Ͻ tomedullary junction reduced MAC to 60% (** P 0.0006) of control Descending Locomotor Command and MAC (all pontine transection data pooled in figure). Mid-medullary transection only reduced MAC to 90% of control (%P Ͻ 0.03). Caudal In particular, we were interested to study the influence of medullary transections at the level of the obex caused MAC to de- the MLR on MAC and isoflurane effects on movement- crease to approximately 50% of control ($ P Ͻ 0.0006) (B) The same related neurons in the MLR that were activated by supra- sagittal template as in (A) depicting the actual range of transection maximal noxious mechanical stimulation. We found that ϭ levels (shaded) in each group, verified histologically. 1 mid-collicu- brainstem transections that encroached on the MLR (i.e., lar transection range; 2 ϭ rostral pons transection range; 3 ϭ pontomedullary transection range; 4 ϭ mid-medullary transection range. mid-collicular transections) caused a small decrease in MAC, Precollicular decerebrations (gray dashed line) and obex-level tran- and transections immediately caudal to the MLR caused sub- sections (black dashed line) were made by viewing the superior stantial decreases in MAC. Moreover, a 32% decrease in colliculus and obex, respectively, but they were not histologically MAC occurred during local MLR inactivation with lido- ϭ ϭ ϭ examined. CN cuneiform nucleus; 7 facial nucleus; Vc tri- caine. We also found that peri-MAC isoflurane significantly geminal subnucleus caudalis. * ϭ significantly different from intact MAC (paired t test). suppresses noxious stimulus-evoked neuronal responses of MLR neurons. Combined spinal depressant and supraspinal MdD contribute to modulating MAC (see proposed facilitatory actions may underlie immobilizing requirements model in fig. 6). We discuss our current findings in the for other anesthetic classes, as this was previously implicated context of the role and relative importance of different for barbiturates16,17 (although tail flick, not MAC, was brainstem regions in modulating isoflurane MAC, effects assessed).

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cranial-bypassed goat studies that trauma appreciably influ- A B 1 12 enced MAC. MAC values of goats recover postbypass, and a

V) 400

µ MAC increase, not decrease, occurs when isoflurane concen- 10 tration to the brain is selectively increased.4 In the latter 300 8 study, Antognini and Borges found that MAC was decreased 6 200 when isoflurane was selectively administered to the torso and 4 ** at low brain concentration (ϳ0.2 MAC), whereas the study 100 * 18 2 by Yang et al. found that MAC was unchanged, but the

Movement Bouts/30 sec Movement separation was limited (lowest achievable isoflurane brain

Peak EMG Amplitude Peak EMG ( 0 0 CON MLR lido Recov Con MLR lido Recov concentration was 0.5 MAC). This difference is more likely

Fig. 4. Mean changes in the amplitude and number of noxious tail explained by low isoflurane concentrations producing an in- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021 clamp-evoked motor responses with 0.6 minimum alveolar concen- crease in supraspinal facilitation that is reversed somewhere tration (MAC) isoflurane before (CON ϭ control), 5–10 min after between 0.2 and 0.5 MAC, rather than trauma. In our cur- lidocaine microinjection into the midbrain locomotor region (MLR rent study, any trauma associated with removal of all fore- lido), and during recovery 20–30 min after MLR lidocaine injection brain structures after precollicular decerebration did not (Recov). Data are shown as mean and SEM. (A) Effect of lidocaine inactivation of the MLR on the mean amplitude of rectified and inte- change MAC nor did complete removal of the cerebellum. grated biceps femoris electromyogram (EMG) activity elicited by su- Furthermore, although MAC decreased after pontomedul- pramaximal noxious tail clamp (30 s). * ϭ significantly different from lary transection, it increased to 90% of control after more control (P Ͻ 0.012; paired t test). (B) Effect of lidocaine inactivation of caudal transections were made. Finally, lidocaine microinjec- the MLR on the mean number of movements elicited by supramaxi- tion into the MLR presumably caused minimal trauma com- mal noxious tail clamp (30 s) during 0.6 and 0.8 MAC isoflurane. ** ϭ significantly different from control (P Ͻ 0.02; paired t test). pared with transections but produced most of the MAC decrease as transections immediately caudal to the MLR. Thus, Peak firing rate of MLR neurons was significantly sup- all these points indicate that MAC changes were primarily pressed by 26% at 0.8 MAC, suggesting that this was perhaps the result of effects on descending modulation and not from a more sensitive measure of sub-MAC motor depression that general trauma-related issues. Another concern relates to the issue of lidocaine spread occurs between 0.4 and 0.8 MAC.6 Effects of isoflurane on outside the MLR. We based our lidocaine concentration and the overall activity of MLR neurons were predictive of MAC, volume on a previous study,19 which found that 0.5 ␮l lido- because responses were significantly decreased only between caine (4%) inactivated a medullary region with a radius of 0.8 and 1.2 MAC, but by 87%. This suggests that isoflurane- 0.5 mm. As we noted, 0.5 ␮l MLR injections decreased induced disfacilitation from the MLR plays a role in the MAC in 50% of animals tested. We then switched to 1 ␮l brainstem-mediated changes that finally lead to immobility. injections, which by spherical volume would inactivate an However, isoflurane suppression of MLR activity is perhaps area with a radius of 0.63 mm. This corresponds well with a necessary, but not sufficient, condition for the final transi- the requirement that both the PPN and the cuneiform nuclei tion to immobility, because we found that mid-medullary (both form the MLR) need to be inactivated to see a consis- transections removed an inhibitory influence on MAC (see tent decrease in MAC. We cannot be certain that lidocaine Descending Nociceptive Modulation and MAC). did not spread to other brain sites that may have influenced The current study was limited in addressing some factors MAC. However, this was unlikely for several reasons. First, to consider. Because noxious stimuli activated MLR neu- we tested a group of animals that received 0.5 ␮l injections rons, isoflurane action in the spinal cord could have indi- dorsal and ventral to the site where we could elicit locomo- rectly affected facilitation from the MLR. In addition, an tion with low-threshold electrical stimulation and never did increase in MAC from descending facilitation does not nec- this affect MAC in any animals. Second, mid-collicular tran- essarily imply that MLR neurons are resistant to isoflurane. sections, which often removed a rostral portion of the MLR, Therefore, it is conceivable that anesthetic effects on mutual minimally reduced MAC by 10%. Furthermore, on the basis interactions between the brainstem and the spinal cord ulti- of our pontine/medullary transection data, lidocaine inacti- mately result in net effects of isoflurane on brainstem or vation of regions caudal to the MLR would remove inhibi- spinal neurons, and MAC being greater in intact compared tion from these areas and thus tend to increase MAC not with spinalized animals. decrease it. On the basis of these findings, it is likely that the One potential concern with the current study and with effect of lidocaine injection on MAC was primarily, if not previous studies on cranial-bypassed goats1,4 is that trauma exclusively, due to inactivation of the MLR. alone might influence MAC. A recent study using emulsified isoflurane in goats avoided potential traumatic effects by Descending Nociceptive Modulation and MAC isoflurane injection into the aorta to achieve a preferential The current data suggest that there is a modulation of body delivery,18 which showed that MAC was unchanged. anesthetic requirements from brainstem sites involved in de- Although data from a nontraumatic preparation are informa- scending nociceptive modulation. Transections ranging tive, there was no evidence in the current study or in previous from the rostral pons (immediately caudal to the MLR) to

Jinks et al. Anesthesiology, V 112 • No 2 • February 2010 322 Brainstem Influences on MAC and Movement Pattern

A Clamp Tail 0.0 MAC Clamp Tail 0.4 MAC 200 Hz

L. L5

R. L5

10 sec Clamp Tail 0.8 MAC Clamp Tail 1.2 MAC Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021 200 Hz

L. L5

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B CD 1400 160 1200 140 IC 1000 120 100 ** CN 800 80 600 PPN 60 + 400 40 200

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* Peak Firing Rate(Hz) 0 0 0.0 0.4 0.8 1.2 0.0 0.4 0.8 1.2 MAC MAC Fig. 5. Effects of isoflurane on supramaximal noxious mechanically evoked responses of midbrain locomotor region (MLR) neurons. Data are shown as mean and SEM. (A) Tail clamp-evoked responses in an MLR neuron (firing rate histogram; bin, 50 ms) that matched movement bouts monitored by recording bilateral L5 ventral root activity (bottom two raw traces). MLR neuronal responses to tail clamp were suppressed by peri–minimum alveolar concentration (MAC) isoflurane. (B) Bar graph showing mean effects of isoflurane on total responses of MLR neurons (n ϭ 11). * Significantly decreased compared with responses under all sub-MAC isoflurane concentrations (P Ͻ 0.001 in all cases). (C) Bar graph showing mean effects of isoflurane on peak firing rate of MLR neurons (n ϭ 11). ** Significantly decreased compared with 0.0 MAC values (P Ͻ 0.034); ϩsignificantly decreased compared with all sub-MAC concentrations (P Ͻ 0.001 in all cases). (D) Sagittal template (lateral 1.9 mm) adapted from Paxinos and Watson31 depicting locations of six histologically identified MLR recording sites. CN ϭ cuneiform nucleus; IC ϭ inferior colliculus; PPN ϭ pedunculopontine nucleus. the pontomedullary junction caused a 40% decrease in creases and increases corresponded with the presence or ab- MAC. These transections left intact the RVM, an area well sence of the medullary level where RVM on and off reside. known to mediate descending facilitatory and inhibitory Transections placed caudal to the RVM (nucleus raphe modulation of nociception (see Refs. 20–23 for reviews). magnus and gigantocellularis pars alpha), but rostral to the The results indicate that when the MLR is compromised, a MdD, caused MAC to increase to near baseline values (com- robust inhibition from the rostral medulla is unmasked that pared with pontine transections). Slightly more caudal tran- is capable of decreasing MAC. This net inhibition could arise sections that removed the MdD caused MAC to drop dra- from the ability of isoflurane to both facilitate nociceptive matically to 50% of control (similar to spinalized animals3). inhibitory “off” cells and inhibit nociceptive facilitatory “on” This suggests that the removal of inhibition from the RVM cells located in the RVM.13 One limitation is that we did not unmasks descending pronociceptive actions from the caudal selectively lesion the RVM, and therefore, other rostral med- medulla. The MdD in the caudal medulla facilitates nocicep- ullary sites could have contributed to MAC changes. How- tive transmission by directly enhancing dorsal horn activity24 ever, the RVM is the medullary component of a part of a and by mandating diffuse noxious inhibitory controls major descending nociceptive modulatory circuit receiving (DNICs). DNIC is a spinal-bulbospinal process through input from the periaqueductal gray. Furthermore, MAC de- which a noxious stimulus at one location suppresses dorsal

Anesthesiology, V 112 • No 2 • February 2010 Jinks et al. PERIOPERATIVE MEDICINE 323

Isoflurane

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+ DH Dorsal Horn

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RVM Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021

+ Ventral Horn BRAINSTEM MN

SPINAL CORD Movement

Fig. 6. Proposed brainstem–spinal cord model depicting sites mediating isoflurane-induced immobility and brainstem influences on minimum alveolar concentration (MAC). “ϩ” represents facilitation, and “Ϫ” represents inhibition. Primary isoflurane effects are shown with a thick arrow, and minor contributions are shown with thin arrows. A noxious stimulus activates primary afferent nociceptors, which in turn activate nociceptive dorsal horn (DH) neurons. Supramaximal noxious stimuli elicit rhythmic, multisegmental locomotor responses by activating inhibitory and excitatory interneurons in the spinal central pattern generating network (CPG), which shapes rhythmic motoneuron (MN) discharge that ultimately leads to organized movement. The black raw tracing in the lower right shows an actual bout of rhythmic hindlimb movement (isometric force) elicited by supramaximal noxious tail clamp. Isoflurane exerts a potent direct action on the ventral spinal cord, where the CPG and motoneurons reside, although the precise contributions from each of these components to immobility is unknown. The main brainstem contribution to the immobilizing requirement of isoflurane is proposed to be derived from a peri-MAC isoflurane-induced reduction in descending facilitation of the spinal locomotor network from the mesencephalic locomotor region (MLR). Isoflurane significantly reduces MLR neuronal responses to noxious stimulation, disfacilitating the CPG (and motoneurons). Smaller contributions to the immobilizing requirement of isoflurane are proposed to be derived from medullary sites mediating descending modulation of the dorsal horn. These include inhibition from the rostral ventromedial medulla (RVM) and facilitation from the medullary dorsal reticular nucleus (MdD). horn neurons in all outlying segments, thereby enhancing Thus, it is possible that these nociceptive modulatory sites nociceptive signal contrast (see Ref. 15 for review). A reduc- play a much larger role in situations that compromise brain- tion in DNIC is thought to contribute to analgesia, as others stem locomotor regions. Although MAC was 90% of control have found morphine to suppresses DNIC in rodents and after mid-medullary transections, movement during sub- humans.25,26 One limitation was that currently we did not MAC isoflurane was nonrepetitive and weaker compared assess the effects of isoflurane on dorsal MdD neurons. How- with conditions in which the MLR was intact. Furthermore, ever, we previously found that DNIC is ablated by isoflurane selective inactivation of the MLR reduced the number and between 0.8 and 1.2 MAC,27 suggesting that isoflurane pro- amplitude of movements at 0.6 MAC (at 0.8 MAC, move- foundly inhibits neuronal activity in the MdD or at least its ment did not occur after lidocaine injection that decreased ability to modulate nociceptive dorsal horn activity. MAC by 32%). Thus, the MLR promotes robust and repetitive movement with isoflurane, conceivably of clinical rele- Relative Importance of MAC-modulating Brainstem Sites vance regardless of MAC changes. to Immobility Our current focus on the MLR was warranted by previous Conclusions studies demonstrating that volatile anesthetics produce immobility mainly by affecting ventral spinal circuitry,8 possi- In summary, we found that the substantially increased bly locomotor networks,6,28 and not by an action on sensory isoflurane immobilizing requirements of the neuraxis-intact dorsal horn neurons.9,27,29–31 Thus, the main brainstem in- or decerebrate versus spinalized animal are due to certain fluence on MAC in intact animals must be derived from regions located in the brainstem. Activity of the MLR likely effects on descending motor commands, more so than from increases MAC requirements and sub-MAC movements descending nociceptive modulation. However, we previously through facilitation of ventral spinal locomotor circuits, found that approximately 10% of isoflurane’s immobilizing whereas more caudal brainstem areas, possibly the RVM and properties seem to be attributed to dorsal horn effects,6 the MdD, may affect the motor response via modulation of which could result from such anesthetic effects on descend- nociceptive dorsal horn activity. Thus, the precise brainstem ing nociceptive modulation. contribution to the immobilizing requirements of isoflurane When the MLR was removed or inactivated, inhibitory seems to result from interplay among descending locomotor influences from the rostral medulla and facilitation from the command and descending nociceptive inhibitory and facili- caudal medulla decreased and increased MAC, respectively. tatory modulation.

Jinks et al. Anesthesiology, V 112 • No 2 • February 2010 324 Brainstem Influences on MAC and Movement Pattern

References 17. Stein C, Morgan MM, Liebeskind JC: Barbiturate-induced inhibition of a spinal nociceptive reflex: Role of GABA 1. Antognini JF, Schwartz K: Exaggerated anesthetic require- mechanisms and descending modulation. Brain Res 1987; ments in the preferentially anesthetized brain. ANESTHESI- 407:307–11 OLOGY 1993; 79:1244–9 18. Yang J, Chai YF, Gong CY, Li GH, Luo N, Luo NF, Liu J: 2. Rampil IJ: Anesthetic potency is not altered after hypother- Further proof that the spinal cord, and not the brain, mic spinal cord transection in rats. ANESTHESIOLOGY 1994; mediates the immobility produced by inhaled anesthetics. 80:606–10 ANESTHESIOLOGY 2009; 110:591–5 3. Jinks SL, Dominguez CL, Antognini JF: Drastic decrease in isoflu- 19. Sandkuhler J, Gebhart GF: Relative contributions of the rane minimum alveolar concentration and limb movement nucleus raphe magnus and adjacent medullary reticular forces after thoracic spinal cooling and chronic spinal transec- formation to the inhibition by stimulation in the periaque- tion in rats. ANESTHESIOLOGY 2005; 102:624–32 ductal gray of a spinal nociceptive reflex in the pentobar- 4. Borges M, Antognini JF: Does the brain influence somatic bital-anesthetized rat. Brain Res 1984; 305:77–87 responses to noxious stimuli during isoflurane anesthesia? 20. Basbaum AI, Fields HL: Endogenous pain control systems: ANESTHESIOLOGY 1994; 81:1511–5 Brainstem spinal pathways and endorphin circuitry. Annu Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/2/316/249507/0000542-201002000-00015.pdf by guest on 27 September 2021 5. Rampil IJ, Mason P, Singh H: Anesthetic potency (MAC) is Rev Neurosci 1984; 7:309–38 independent of forebrain structures in the rat. ANESTHESI- 21. Mason P, Leung CG: Physiological functions of pontomed- OLOGY 1993; 78:707–12 ullary raphe and medial reticular neurons. Prog Brain Res 6. Jinks SL, Bravo M, Hayes SG: Volatile anesthetic effects on 1996; 107:269–82 midbrain-elicited locomotion suggest that the locomotor 22. Gebhart GF, Sandkuhler J, Thalhammer JG, Zimmermann network in the ventral spinal cord is the primary site for M: Inhibition of spinal nociceptive information by stimu- immobility. ANESTHESIOLOGY 2008; 108:1016–24 lation in midbrain of the cat is blocked by lidocaine mi- 7. Whelan PJ: Control of locomotion in the decerebrate cat. croinjected in nucleus raphe magnus and medullary retic- Prog Neurobiol 1996; 49:481–515 ular formation. J Neurophysiol 1983; 50:1446–59 8. Kim J, Yao A, Atherley R, Carstens E, Jinks SL, Antognini JF: 23. Heinricher MM, Tavares I, Leith JL, Lumb BM: Descending Neurons in the ventral spinal cord are more depressed by control of nociception: Specificity, recruitment and plas- isoflurane, halothane, and propofol than are neurons in ticity. Brain Res Rev 2009; 60:214–25 the dorsal spinal cord. Anesth Analg 2007; 105:1020–6 24. Dugast C, Almeida A, Lima D: The medullary dorsal retic- 9. You HJ, Colpaert FC, Arendt-Nielsen L: Nociceptive spinal ular nucleus enhances the responsiveness of spinal noci- withdrawal reflexes but not spinal dorsal horn wide-dy- ceptive neurons to peripheral stimulation in the rat. Eur namic range neuron activities are specifically inhibited by J Neurosci 2003; 18:580–8 halothane anaesthesia in spinalized rats. Eur J Neurosci 25. Le Bars D, Chitour D, Kraus E, Clot AM, Dickenson AH, 2005; 22:354–60 Besson JM: The effect of systemic morphine upon diffuse 10. Antognini JF, Atherley RJ, Dutton RC, Laster MJ, Eger EI II, noxious inhibitory controls (DNIC) in the rat: Evidence for Carstens E: The excitatory and inhibitory effects of nitrous a lifting of certain descending inhibitory controls of dorsal oxide on spinal neuronal responses to noxious stimula- horn convergent neurones. Brain Res 1981; 215:257–74 tion. Anesth Analg 2007; 104:829–35 26. Le BD, Willer JC, De BT: Morphine blocks descending pain 11. Carlson JD, Selden NR, Heinricher MM: Nocifensive reflex- inhibitory controls in humans. Pain 1992; 48:13–20 related on- and off-cells in the pedunculopontine tegmen- 27. Jinks SL, Antognini JF, Carstens E: Isoflurane depresses tal nucleus, cuneiform nucleus, and lateral dorsal tegmen- diffuse noxious inhibitory controls in rats between 0.8 and tal nucleus. Brain Res 2005; 1063:187–94 1.2 minimum alveolar anesthetic concentration. Anesth 12. Skinner RD, Garcia-Rill E: The mesencephalic locomotor Analg 2003; 97:111–6 region (MLR) in the rat. Brain Res 1984; 323:385–9 28. Jinks SL, Atherley RJ, Dominguez CL, Sigvardt KA, Antog- 13. Jinks SL, Carstens E, Antognini JF: Isoflurane differentially nini JF: Isoflurane disrupts central pattern generator activ- modulates medullary on and off neurons while suppress- ity and coordination in the lamprey isolated spinal cord. ing hind-limb motor withdrawals. ANESTHESIOLOGY 2004; ANESTHESIOLOGY 2005; 103:567–75 100:1224–34 29. Barter LS, Mark LO, Jinks SL, Carstens EE, Antognini JF: 14. Lima D, Almeida A: The medullary dorsal reticular nucleus Immobilizing doses of halothane, isoflurane or propofol, as a pronociceptive centre of the pain control system. do not preferentially depress noxious heat-evoked re- Prog Neurobiol 2002; 66:81–108 sponses of rat lumbar dorsal horn neurons with ascending 15. Le BD, Villanueva L, Bouhassira D, Willer JC: Diffuse nox- projections. Anesth Analg 2008; 106:985–90 ious inhibitory controls (DNIC) in animals and in man. 30. Jinks SL, Martin JT, Carstens E, Jung SW, Antognini JF: Peri-MAC Patol Fiziol Eksp Ter 1992:55–65 depression of a nociceptive withdrawal reflex is accompanied 16. Morgan MM, Stein C, Liebeskind JC: Intrathecal adminis- by reduced dorsal horn activity with halothane but not isoflu- tration of pentobarbital and THIP: Evidence for descend- rane. ANESTHESIOLOGY 2003; 98:1128–38 ing excitatory and inhibitory modulation of a spinal reflex. 31. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coor- Proc West Pharmacol Soc 1987; 30:249–51 dinates, 4th Edition. New York, Academic Press, 1998

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