Influence of chloralose on brain regional glucose utilization

Brain Research, 233 (1982) 173-180

173

Elsevier Biomedical Press

I N F L U E N C E OF C H L O R A L O S E ON BRAIN R E G I O N A L G L U C O S E UTILIZATION

ROBERT E. DUDLEY, STANLEY R. NELSON* and FRED SAMSON Departments of Pharmacology, Anatomy and Ralph L. Smith Research Center, The University of Kansas, College of Health Sciences and Hospital, Kansas City, KS 66103 (U.S.A.)

(Accepted July 9th, 1981) Key words: chloralose - - brain regional glucose use - - anesthetics - - 2-deoxyglucose

SUMMARY Choralose, a widely used anesthetic in neurophysiology, produces a unique pattern of anesthesia characterized by both an excitant (myoclonic jerks and startle response) and depressant (sedation and anesthesia) action. We investigated the influence of chloralose on the rate of regional brain glucose metabolism to determine if chloralose produces anesthesia by hyperexciting certain brain regions. That is, does chloralose act as an 'epileptoid anesthetic'. Rats were anesthetized with either 60 or 120 mg/kg chloralose and regional brain glucose utilization rates quantitated by the 2deoxyglucose method. In chloralose-anesthesized rats, glucose consumption rates decreased in the frontal and auditory cortex, reticular nucleus of thalamus, superior colliculus, medial geniculate body, midbrain reticular formation and hippocampus. Rates of glucose use were not decreased in the lateral lemniscus and a zone in the vicinity of the oculomotor nucleus, medial longitudinal fasciculus and surrounding reticular formation. Since chloralose did not induce any discernible focal points of high activity, chloralose appears not to be an epileptogen. Rather, chloralose appears to act as a general depressant except in certain gray areas of the midbrain and lower brain stem. Retained and possibly increased functional activity in the vicinity of the oculomotor nucleus and medial longitudinal fasciculus may represent active reflex pathways involved in mediating the paradoxical startle response and myoclonic activity observed in chloralose-anesthetized animals.

INTRODUCTION Alpha-chloralose is a widely used anesthetic agent in neurophysiological re* To whom correspondence should be addressed at: Department of Anatomy, The University of Kansas, College of Health Sciences and Hospital, Kansas City, KS 66103, U.S.A. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

174 search, but the mechanisms of action of this drug remain unclearL Initial findings suggested that chlo~ alose acts as a convulsant as well as an anesthetic". Evidence for its convulsant action are spontaneous myoclonic jerks in response to stimulation (e.g. noise, touch); whereas the depressant action is evident from sedation and anesthesia. Additional evidence for a dual action of chloralose comes from neurophysiological studies such as those of Winters and co-workers from which they concluded that chlo~alose does not meet the criteria necessary to be classified as an anesthetic 17. Rather, they postulate that chloralose hyperexcites certain brain areas and the responses of chloralose-treated animals more closely mimic an 'epileptoid state' rather than anesthesia. The 2-deoxyglucose (2-DG) functional mapping method was used to quantitate regional brain glucose use rates in chloralose-anesthetized rats 1,5. This method has the advantage in that a broad pictorial view of regional brain activity can be obtained, and there is good evidence that the rate of glucose use is closely coupled to the local functional activity in the brain3,13,lS, 16. We employed this procedure to determine if certain regions of brain a~e metabolically activated under chloralose anesthesia and, if so, whether such findings support the view that chloralose is an 'epileptogen '18. Our results show that chloralose depresses most areas of the CNS. Exceptions to this were segments of the caudal portion of the auditory system and possible activation in a zone adjacent to and possibly including the oculomotor nucleus, midbrain reticular formation and medial longitudinal fasciculus. Thus, our findings do not support the view that chloralose is an 'epileptogen' in the manner proposed by Winters and Spooner is. Rather, chloralose produced a unique anesthetic pattern different from that of barbiturates 15 and not at all like that o f the epileptogen, ketaminelL MATERIALS AND METHODS Fourteen male Wistar rats (Charles River), 8-12 weeks old were used. Control rats (n .... 5) were injected intraperitoneally (i.p.) with propylene glycol (0.25 ml), the vehicle fol chloralose. Animals in the two experimental groups were injected with eithel 60 (n -- 4) or 120 mg/kg (n == 5) of chloralose, respectively. Crystalline achloralose (Sigma Chemical Co., St. Louis, MO) which contained 10 ~o of the fl-isomer was used. However, Monroe et al. 9 have shown that the a-isomer has both the anesthetic and excitatory properties while the fl form is relatively inactive. Chloralose was dissolved in propylene glycol on the day of use to minimize conversion to the/3 isomer of the drug. Prior to drug injections, indwelling polyethylene catheters were placed in the femoral artery and vein of each rat so that 2-deoxyglucose (2-DG) could be injected into the vein and serial blood samples taken from the artery during the experiment. Surgery for catheter placement was done under halothane anesthesia, 2 ',~,i halothane with 100~o oxygen. Chloralose was injected 2-3 h after the animals recovered from anesthesia. Regional biain glucose use was determined following the method of Sokoloff et al. aS. [laC]2-Deoxyglocuse, 100 #Ci/kg, was injected intravenously into rats 45 min after rats received either propylene glycol or chloralose. Immediately after 2-DG was

175 injected, serial arterial blood samples were collected over the next 45 min. Blood decay curves of 2-DG and plasma glucose levels were determined from these samples. Fortyfive min after 2-DG was injected, rats were decapitated and brains removed and frozen in Freon-12 at --70 °C. Brains were sectioned at 20 # m and the sections dried on glass coverslips at 60 °C. Sections were placed on Kodak Min-R-X-ray film along with [14C]methyl methacrylate standards and allowed to expose the film for 10 days. Optical densities of brain regions were obtained from autoradiographs with a densitometer (Model TBX, Tobias Associates, Inc., Ivyland, PA) with a 0.3 mm aperature. The rate of regional glucose utilization was determined from the optical density values, 2-DG decay curve and plasma glucose concentration using the operational equation of the method ~5. In order to identify specific anatomical structures seen on autoradiographs, Luxol Fast Blue-stained sections at equivalent brain levels were made from rats that had not previously undergone experimental procedures. These were carefully matched with the autoradiographs and structures were subsequently identified from a stereotaxic rat atlas~L Statistical differences in regional brain glucose use between the groups of animals was determined by a one way analysis of variance and Duncan's multiple range test ~. RESULTS A measme of responsiveness during chloralose anesthesia was determined by treating rats with various doses of chloralose and obselving them for righting reflex, pain and response to noise (startle response). Doses of 60 and 90 mg/kg caused a loss of the righting reflex but the time for tail withdrawal from a 55 °C water bath 7 was not increased. With 120 mg/kg, the response of the rat to pain was depressed as indicated by an increase in the time for tail withdrawal from 55 °C water, from 2 to 11 s (P < 0.01). In contrast to depression of these reflexes, rats treated at all 3 doses of chloralose showed sudden jerking of the extremities in response to a sharp, loud noise (startle response). Chloralose anesthesia is unique in accentuating responses to noise even when righting reflex and pain responses are depressed. The functional activity of brain regions was studied at doses of chloralose (60 and 120 mg/kg) which produced two levels of anesthesia, respectively. The shades of gray in autoradiographs from control and chloralose-treated rats give a relative, qualitative measure of glucose use in the brain (Fig. 1). As shown previously 15, the brain regions in control rats showing high rates of glucose use were: inferior colliculus, auditory cortex, thalamus and lateral lemniscus. In the chloralose-treated rats, a midbrain density not seen on the control autoradiographs is apparent, but, other than this there appear to be no discrete, dense areas indicative of epileptic foci. Quantitative measure of the glucose use in control and chloralose treated rats was determined for 10 areas of bIain. Eight of these areas had significant decreases in glucose use at one or both levels of chloralose treatment (Fig. 2). The two areas which clearly did not show a reduction in glucose were the lateral lemniscus and an area in the vicinity of the oculomotor nucleus, medial longitudinal fasciculus (MLF) and

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Fig. 1. Representative autoradiographs from control rats and from rats treated with a-chloralose, 60 mg/kg. Rats were injected with 14C-labeled 2-deoxyglucose 30 min after receiving either propylene glycol or a-chloralose. From top to bottom, brain sections were taken from the level of the medial geniculate body, inferior colliculus and medulla. Abbrevations: SC, superior colliculus; MG, medial geniculate; MLF, medial longitudinal fasciculus and surrounding area; IC, inferior colliculus; RF, reticular formation; LL, lateral lemniscus. midbrain reticular formation (Figs. 2 and 3). It should be noted that for reasons not clear to us, the glucose utilization rates determined for various brain regions in our control rats are lower than those observed by otherslk Some o f this difference may be due to variability in laboratory conditions such as light and sound levels during the experiments. Identification o f the activated midbrain region in close proximity to the M L F was attempted by measuring the area corresponding to the M L F on autoradiographs and comparing this area with the area on Luxol Fast Blue-stained sections (Fig. 3): Measurements were made with a disc micrometer in a light microscope. The area identified as the M L F on autoradiographs (0.45 mmZ) was 20 times larger than the

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Fig. 2. Regional glucose use in control and chloralose-anesthetized rats. The horizontal bars represent the mean rate of glucose use in control rats (C) and in rats which received 60 mg/kg (60) and 120 mg/kg (120) of chloralose. The number of rats in each group C, 60 and 120, is 5, 4 and 5, respectively. Narrow horizontal bars indicate S.E. *P < 0.05. corresponding area on stained sections (0.02 ram2). S o m e of this difference can be accounted for by the o c u l o m o t o r nucleus which lies dorsal to the M L F , but no discrete anatomical structures could be identified ventrally. Further, the activated areas were observed to m o v e m o r e caudally than could be accounted for solely by an increased activity in the o c u l o m o t o r nucleus. This implicates involvement of a portion o f the medial reticular formation in the responses seen to chloralose. DISCUSSION A noteworthy characteristic o f chloralose anesthesia is the induction o f m y o clonic m o t o r m o v e m e n t s and the exaggerated responses to sudden stimuli 6,9. The neuronal circuitry o f these excitatory responses has been studied, largely by electrical recordings, and the cortical m o t o r areas and pyramidal tracts appear to mediate the clonic response to sudden stimuli 1. Although the cortical activity generated during chloralose anesthesia is hypersynchronous 4 this overall level o f cortical activity is not apparent from these studies. Our estimates o f the rate o f cortical glucose use s h o w that

178

Fig. 3. Photomicrograph of Luxol Fast Blue-stained section of rat midbrain showing relationship between the medial longitudinal fasciculus (A), oculomotor nucleus (B), red nucleus (C) and cerebral aqueduct (D). Magnification is 40-:. The increased size and intensity in the medial longitudinal fascicular zone as seen in the autoradiographs of chloralose-treated rat brains (Fig. l) may be accounted for by increased activity occurring in the medial portions of the reticular formation adjacent to the medial longitudinal fasciculus as well as likely involvement of the oculomotor nucleus.

179 the average cortical activity is reduced. One of the cortical components with reduced activity may involve cortical inhibitory mechanisms which Jung believes are responsible for the loss of normal cortical rhythm during chloralose anesthesia s. The electrical activity of the reticular formation is sensitive to various anesthetics 10 and may be an important structure in producing an anesthetic state. We found the overall rate of glucose use in the reticular formation decreased, indicating a reduction in activity. However, there was retained activity at or above control levels in the vicinity of the oculomotor nucleus, M L F and surrounding reticular formation (Figs. 1 and 3). In rat brain sections stained with Luxol Fast Blue, the M L F is a small group of myelinated fibers at the ventral border of the periaqueductal gray matter (Fig. 3), whereas the area with normal or possibly increased glucose use extends beyond the limits of the M L F and appears to involve a small, medial segment of the reticular formation. The dense zone is well circumscribed with no indication of lateral spread into surrounding tissue or activation of brain stem nuclei as one might expect were this an epileptic focus. It should be noted that the dense region near the M L F may be more apparent on autoradiographs because of reduced density in surrounding brain stem. Also of importance is the caudal extent of the increased midline activity beyond the oculomotor nucleus. This observation suggests that the M L F and surrounding reticular formation may be unaffected or possibly activated by chloralose and may be involved in mediating reflex pathways in chloralose anesthetized rats. Shimamura and YamauchP 4 observed increased electrical activity (excitatory in nature) in discrete midline structures in brain stems of cats anesthetized with choloralose --- a finding which correlates well with the increased glucose utilization observed in similar regions of choloralose-anesthetized rats of our study, We believe the increased regions of glucose use seen in midline regions of the brain stem and midbrain of rats in our study may be evidence for rostral extension of the chloralose reflex pathway proposed by Shimamura and YamauchP4. The pattern of regional metabolic changes found with chloralose differs markedly from that of ketamine u. This supports the view of Winters et al. that, 'the concept of a single anesthetic state is not valid uT. On the other hand, the findings do not support the view that chloralose produces a loss of arousal secondary to formation of epileptic focP 8. Although the rate of glucose use of most gray matter was depressed, the caudal auditory nuclei and a zone of midbrain surrounding the M L F was maintained at or above control levels. This was in contrast to the findings in rats anesthetized with a known convulsant anesthetic, ketamine, which markedly activates neurons along the hippocampal fissure and depiesses the inferior colliculus u. We suggest that chloralose is not an epileptogenic anesthetic, at least not in as clear a manner as ketamine. Its anesthetic action appears to result from a depression of regions of gray matter, but some primitive brain stem sensory-motor systems remain functional. The unusual activity in the neighborhood of the oculomotor nucleus and M L F is probably involved in the set of characteristics peculiar to choloralose anesthesia (i.e. startle response).

180 ACKNOWLEDGEMENTS S u p p o r t in part by U S P H S G r a n t H D 02528 a n d U.S. A r m y Med. C o m m . D A M D 17-78-C-8039. We t h a n k Dr. F l o y d Foltz for his help in identifying a n a t o m i c a l structmes a n d wish to acknowledge the expert assistance of R o b e r t S. Cross.

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3 Collins, R. C., Kennedy, C., Sokoloff, L. and Plum, F., Metabolic anatomy of focal motor seizures, Arch. Neurol., 33 (1976) 536-542. 4 Domino, E. F., and Ueki, S., An analysis of the electrical burst phenomenon in some rhinencephal ic structures of the dog and monkey, Electroenceph. clin. NeuropshyioL, 12 (1960) 635-648. 5 Duncan, B., Multiple range and multiple Ftests, Biometrics, 11 (1955) 1-42. 6 Hanriot, M. and Richer, C., The chloraloses (Les chloraloses), Arch. int. Pharmacodyn., 3 (1897) 191-211. 7 Jansen, P. A. J., Niemegeers, C. J. E. and Dony, J. G. H., The inhibitory effect of fentanyl and other morphinetike analgesics on the warm water induced tail withdrawal reflex in rats, Arzneim.Forsch., 13 (1963) 502-507. 8 Jung, R. In J. F. Dehresnaye (Ed.), Brain Mechanisms and Conciousness, Charles C. Thomas, Springfield, 1954, p. 198. 9 Monroe, R. R., Balis, G. U. and Ebersberger, E., The hypnotic effects of a!pha and beta chloralose in rats, Curt. Ther. Res., 5 (1963) 141-153. 10 Mori, K., Winters, W. D. and Spooner, C. E., Comparison of reticular and cochlear multiple unit activity with auditory evoked responses during various stages induced by anesthetic agents. 11, Electroenceph. elin. NeurophysioL, 24 (1968) 242-248. 11 Nelson, S. R., Howard, R. B., Cross, B. S. and Samson, F. E., Ketamine induced changes in regional glucose utilization in the rat brain, Anesthesiology, 52 (1980) 28-32. 12 Pelligrino, L. J. and Cushman, A. A., A Stereotaxic Atlas of the Rat Brain, Appleton-CenturyCrofts, New York, 1967. 13 Sharp, F. R., Kauer, J. S. and Shepherd, G. M., Local sites of activity related glucose metabolism in rats olfactory bulb during olfactory stimulation, Brain Research, 98 (1975) 596-600. 14 Shimamura, M. and Yamauchi, T., The neural mechanisms of the chloralose jerk with special reference to its relationship with the spino-bulbo spinal reflex, ,lap. J. Physiol., 17 (1967) 738-745. 15 Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. and Shinohara, M., The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthesized albino rat, J. Neurochem., 28 (1977) 897-916. 16 Sokoloff, L., The relationship between function and energy metabolism; its use in the localization of functional activity in the nervous system, Neurosci. Res. Bull., 19 (1981) 159-210. 17 Winters, W. D., Ferrar-Allado, T., Guzman-Flores, C. and Alcazar, M., The cataleptic state induced by ketamine: a review of the neuropharmacology of anesthesia, Neuropharmacology, 11 (1972) 303-315. 18 Winters, W. D. and Spooner, C. E., A neurophysiological comparison of alpha-chloralose and gamma-hydroxybutyrate in cats, Electroenceph. clin. Neurophysiol., 20 (1966) 83-90.