Supraspinal, Not Spinal, α₂ Adrenoceptors Are Involved in the Anesthetic-Sparing and Hemodynamic-Stabilizing Effects of Systemic Clonidine in Rats

Abstract

Clonidine, an α₂ agonist, reduces the anesthetic requirement and attenuates harmful hemodynamic responses to noxious stimuli. We examined the responsible sites of action in the central nervous system for the minimum alveolar anesthetic concentration (MAC) and MAC blocking adrenergic response (MAC-BAR) reducing effects of systemically administered clonidine in halothane-anesthetized rats. The MAC for halothane was determined by the tail clamp method, and MAC-BAR was defined as the MAC which attenuated hemodynamic responses within 10% after the tail clamp. We examined the effect of IV clonidine in the presence of rauwolscine, an α₂ antagonist given through IV, intrathecal (IT), intracisternal (IC), or intracerebroventrical (ICV) routes. IV clonidine reduced MAC and MAC-BAR dose-dependently. IV and ICV rauwolscine antagonized the MAC-reducing effect of clonidine, whereas IC and IT rauwolscine did not. In comparison, IV, ICV, and IC rauwolscine antagonized the MAC-BAR-reducing effect of clonidine; IT rauwolscine had no effect. Our data demonstrate that the α₂ adrenoceptors in the regions above mesencephalon and both the regions above mesencephalon and the lower brainstem are responsible for the MAC and MAC-BAR-reducing effect of systemic clonidine in rats, respectively. However, the spinal α₂ adrenoceptors were not involved in these effects of clonidine.

Implications: 

In the regions above mesencephalon, α₂ adrenoceptors were the most responsible for the minimum alveolar concentration-reducing effect and both the lower brainstem and regions above mesencephalon were involved in the minimum alveolar concentration blocking adrenergic response-reducing effect of clonidine. The spinal α₂ adrenoceptors did not significantly contribute to these effects of clonidine.

The α₂ adrenoceptors are widely distributed in the central nervous system (CNS), and activation of these receptors produces several beneficial functions, including analgesia, minimum alveolar concentration (MAC)-reducing action, and hemodynamic stability (^1,2). Sites of α₂ adrenoceptors responsible for the analgesic action of α₂ agonists involve both supraspinal and spinal mechanisms (³). However, the precise sites in the CNS, the MAC-reducing action, and MAC-blocking adrenergic response (MAC-BAR) of α₂ agonists are obscure.

The spinal cord may be involved with the anesthetic action of volatile anesthetics (^4,5) and spinal administration of morphine and clonidine may reduce MAC in both animals and humans (^6,7). Thus, the spinal cord may play an important role in the MAC-reducing effect of α₂ agonists, although this idea is not universally accepted (^8,9). Clonidine is an α₂ agonist, which has been most widely applied to clinical anesthesia and systemic administration, via the IV, IM, transdermal, and oral routes. It is usually used in clinical situations (^1,2). The current study was designed to clarify the responsible sites of α₂ adrenoceptors for the MAC and MAC-BAR-reducing effects of systemically administered clonidine in rats.

Methods

Our experiments were performed according to the protocol approved by the Animal Care Committee of Osaka University, Faculty of Medicine. Male Sprague-Dawley rats weighing 280–440g were housed in a temperature controlled environment. Lighting was on a 12/12 h light/dark cycle at 8:00–20:00 h for at least 1 wk with food and water available ad libitum. All experiments were performed between 10:00 and 16:00 h to avoid the influence of circadian variation on anesthetic requirements (¹⁰).

The animals were anesthetized with 2.0% halothane (vol) in 100% oxygen in a 60-l glass chamber. After loss of righting reflex, 2.0% halothane was administered via face mask, and then, a tracheotomy was performed. The lungs were mechanically ventilated with a tidal volume of 10 mL/kg at a rate of 40–50 breaths/min. Then, the inspired halothane concentration was maintained at 1.0%, which was monitored continuously with an infrared gas analyzer and intermittently with a mass spectrometer. For blood pressure monitoring and drug administration, 24-gauge catheters were inserted into the femoral artery and the subclavian vein, respectively. Throughout the experiments, the rectal temperature was maintained at 37° ± 0.5°C with a heating pad.

MAC for halothane was determined by using the tail-clamp method (¹¹). The tail of each rat was clamped with a full length hemostat (20 cm) to the first ratchet position for 60 s, and the rats were observed for the presence or absence of withdrawal movement. When withdrawal movements were elicited, the inspired concentration of halothane was increased by 10%, and reduced by 10%, when such movement was absent. The halothane concentration was maintained for 20 min, and the tail was reclamped. We defined MAC as the median concentration of halothane between the largest concentration with movement and the smallest concentration without movement. After determination of MAC for halothane, the halothane concentration was maintained within 1.4% for 30 min, and the MAC-BAR of halothane was determined by using the tail clamp method. MAC-BAR was defined as the median concentration of halothane between the largest concentration with hemodynamic responses and the smallest concentration without hemodynamic responses (blood pressure and heart rate) defined that an increase of 10% or more from predrug administration value to during the tail clamp.

We determined MAC and MAC-BAR of halothane in the following groups:

• 1. Control group (n = 8): the vehicle (0.5 mL) was administered IV 30 min before the first clamp;
• 2. Clonidine group (n = 24): 10 (n = 8), 30 (n = 8), and 100 (n = 8) μg/kg of clonidine was administered as in the control group, respectively; and
• 3. Rauwolscine group (n = 32): rauwolscine, an α₂ antagonist was coadministered with 100 μg/kg of IV clonidine. Rauwolscine was given through four different routes; that is, by IV, intrathecal (IT), intracisternal (IC), or intracerebroventrical (ICV) routes. The dose tested of IV rauwolscine was 20 mg/kg and the dose of IT, IC, and ICV rauwolscine was 40 μg/kg.

The IT catheters were implanted into the spinal cavity 8.5 cm caudally through the atlantooccipital membrane (^12,13). During the recovery period, rats demonstrating weakness or paresis were excluded from the experiment. At the seventh postoperative day, 1.0% lidocaine (10 μL) was injected through the catheter, and motor weakness of the lower extremities was checked to confirm the optimal position of the catheter. At the tenth day postoperatively, the experiment was performed. Rauwolscine, in a volume of 10 μL, was injected through the implanted catheter followed by the same volume of physiologic saline to flush the catheter. For IC administration, we opened the occipital skin of the rats and exposed the atlantooccipital membrane. Then, drugs were directly injected into the cisterna magna through this membrane by using a 30-gauge needle. For ICV administration, we exposed the temporal skull and drilled a hole in the skull. Then, a 30-gauge needle was inserted stereotaxically into the lateral ventricle according to the atlas of the rat brain (¹⁴). After cannulation, 7 days were allowed for animal recovery. For IC and ICV administration, 10 μL of rauwolscine was given over 2 min to minimize tissue disruption. After the experiments, the rats were killed with a large dose of pentobarbital, and 10 μL of dye was injected to confirm the distribution of the drug. All data from multiple groups were expressed as mean ± SD and analyzed by one-way analysis of variance followed by Scheffé’s test. Significance was defined as P < 0.05.

Results

The mean arterial pressure in the IV rauwolscine group was significantly lower; however, heart rate was not different between groups (Table 1). Clonidine reduced the MAC and MAC-BAR of halothane in a dose-dependent fashion to a maximum of 30% and 37%, respectively (Fig. 1). IV and ICV rauwolscine antagonized the effect of clonidine on MAC, whereas IC and IT rauwolscine did not affect the MAC-reducing effect of clonidine (Fig. 2). In comparison, IV, ICV, and IC rauwolscine antagonized the effect of clonidine on MAC-BAR, although IT rauwolscine had no effect on the MAC-BAR-reducing effect of clonidine (Fig. 3).

Table 1: Hemodynamic Data Before the First Clamp During Halothane Anesthesia

Figure 1: The effect of IV clonidine on minimum alveolar anesthetic concentration (MAC) and MAC-blocking adrenergic response (MAC-BAR) of halothane anesthesia in rats. ▩ MAC; MAC-BAR. Values are mean ± SD. *P < 0.05 compared with vehicle.

Figure 2: The effect of IV, intracisternal, lateral ventricle and intrathecal rauwolscine on the minimum alveolar anesthetic concentration (MAC)-sparing effect of systemic clonidine for halothane anesthesia in rats. Values are mean ± SD. *P < 0.05 compared with vehicle.

Figure 3: The effect of IV, intracisternal, lateral ventricle and intrathecal rauwolscine on minimum alveolar anesthetic concentration-blocking adrenergic response (MAC-BAR)-sparing effect of systemic clonidine for halothane anesthesia in rats. MAC-BAR. Value are mean ± SD; *P < 0.05 compared with vehicle.

ICV dye was confirmed on the surface of the lateral ventricle and the ventricle of the medulla oblongata; the dye at the lateral ventricle was more concentrated. In addition, the dye was not seen on the spinal cord. IC dye was confirmed on the ventricle surface of the medulla oblongata, however, not on the spinal cord. IT dye was confirmed only on the lower spinal cord. Our results were similar to the previous report by Szabo and Urban (¹⁵) who demonstrated that 25 μL/kg ICV injection of dye in rabbits stained the ventral surface of the medulla oblongata and pons, not the spinal cord.

Discussion

We conclude that the MAC-reducing effect of systemic clonidine is reversed by IV and ICV rauwolscine, not by IC and IT rauwolscine, suggesting that the region above mesencephalon is responsible for the MAC-reducing effect of systemic clonidine. In comparison, the MAC-BAR-reducing effect of systemic clonidine was reversed by IV, ICV, and IC rauwolscine, whereas IT rauwolscine did not affect the MAC-BAR-reducing effect of clonidine, suggesting that the supraspinal region is responsible for the MAC-BAR-reducing effect of systemic clonidine.

The different routes of drug administration may be helpful in elucidating the site of action for a particular response. Although centrally administered rauwolscine may be distributed outside the CNS, a smaller dose of central rauwolscine was equally effective to systemic rauwolscine, suggesting a central site of action. To further elucidate the responsible site in the CNS for the MAC and MAC-BAR-reducing effects of clonidine, we administered rauwolscine through three different routes: IT, IC and ICV. The cerebrospinal fluid flowing from regions above mesencephalon to the spinal cord may affect the distribution of the drug in the CNS (¹⁶). However, the drug is diluted so the concentration of the drug may be smaller at the injected site. Our postexperiment study using dye confirmed this. Thus, although ICV administration of rauwolscine may be transported from the lateral ventricle to the lower brainstem and the spinal cord, ICV rauwolscine may be most effective above mesencephalon rather than at the lower brainstem and spinal cord. Similarly, IC and IT rauwolscine may work most effectively at the lower brainstem and spinal cord, respectively.

Because previous studies have demonstrated that IT clonidine results in hemodynamic stabilization and antinociceptive effects (^8,17), we hypothesized that the spinal cord plays an important role in the MAC and MAC-BAR sparing effect of clonidine. Thus, that IT rauwolscine did not antagonize the MAC or MAC-BAR-reducing effect of clonidine was unexpected.

Perhaps the route of the administration of clonidine can explain our results. Clonidine given IT produces significant analgesia (¹⁷). However, we gave clonidine systemically. The MAC and MAC-BAR-reducing effect of systemic clonidine may be more dependent on the supraspinal α₂ adrenoceptors than spinal α₂ adrenoceptors. The second possible explanation may be because of the dose of clonidine we administered. Pertovaara et al. (³) reported medetomidine, a selective α₂ agonist, suppressed nociceptive sensory neuronal and reflex responses because of a spinal site of action with a large dose (anesthetic dose) and a supraspinal site of action with a small dose (subanesthetic, sedative dose), respectively, in rats. The dose of clonidine we used was subanesthetic, so the supraspinal site of action may be more predominant than the spinal site of action in our study. The third explanation is that the MAC and MAC-BAR-reducing effect of clonidine is not mediated at the level of the spinal cord or at least not in the immediate subarachnoid area. Therefore, when α₂ agonists are administered systemically, spinal rauwolscine did not significantly affect the MAC and MAC-BAR-reducing effects of clonidine. Our data cannot exclude involvement of spinal α₂ adrenoceptors in the MAC and MAC-BAR-reducing effects of clonidine. In fact, there are several animal studies documenting that spinal α₂ agonists affect MAC and MAC-BAR (^8,9).

Another interesting finding of this study is that the sites responsible for the MAC and the MAC-BAR-reducing effects may be different. Our finding that the ICV, not IC, rauwolscine reversed the MAC-reducing effect suggests that the rostral brain, or regions above mesencephalon, are the most likely sites of clonidine’s effect. In comparison, both ICV and IC rauwolscine reversed the MAC-BAR-reducing effect of clonidine, suggesting that the lower brainstem, as well as the regions above mesencephalon, may be involved in the MAC-BAR-reducing effect of clonidine. Previous studies indicate that α₂ adrenoceptors are concentrated in the rostroventrolateral medulla (RVLM). In addition, adrenergic neurons in the C1 area of the RVLM project to excitatory sympathetic paraganglionic neurons of the spinal cord and nucleus tractus solitarii, which is functionally connected with the C1 area to control the sympathovagal activity (^18–20). Collectively, RVLM is an important site for sympathovagal activity and cardiovascular homeostasis. Therefore, activation of α₂ adrenoceptors in the RVLM may contribute to the MAC-BAR-reducing effect of clonidine. In comparison, our data suggest that the sympatholytic modulation in the RVLM is not significantly involved in the MAC-reducing-effect of systemically administered clonidine. We cannot identify the precise location of α₂ adrenoceptors in the regions above mesencephalon involved in the MAC-reducing-effect of clonidine. The α₂ adrenoceptors are widely distributed in the regions above mesencephalon, including the cerebral cortex, thalamus, and hypothalamus (²¹). A previous report documented that systemic medetomidine significantly suppressed the nociceptive responses at the thalamic level (³), which might play a critical role in the MAC-reducing-effect of clonidine.

Rampil et al. (^4,22) reported that the lower brainstem and spinal cord are responsible for purposeful movement against noxious stimuli in rats anesthetized with isoflurane. Furthermore, Antognini and Berg (²³) documented that the brain has little effect on MAC-BAR for isoflurane in goats investigated with selective blood perfusion to either head or torso. On the contrary, our data suggest that the supraspinal, not the spinal, region is important for the MAC and MAC-BAR-reducing effect of systemic clonidine. Presumably, activation of supraspinal α₂ adrenoceptors may induce the exogenous modulation of MAC and MAC-BAR through the descending neural pathway.

In conclusion, the MAC-reducing-effect of systemically administered clonidine may be mediated through α₂ adrenoceptors in the regions above mesencephalon in rats, whereas both the regions above mesencephalon and lower brainstem are responsible for the MAC-BAR-reducing effect of clonidine. Spinal α₂ adrenoceptors may not be involved in these effects.