Journal of Neuroscience Methods 77 (1997) 49 53

A simple method, using 2-hydroxypropyl-i-cyclodextrin, of administering h-chloralose at room temperature

R. James Storer a, Paul Butler b, Karen L. Hoskin a, Peter J. Goadsby a,*
a

Institute of Neurology, The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK b Pzer Central Research Limited, Sandwich, Kent, UK Received 5 March 1997; received in revised form 29 May 1997; accepted 9 June 1997

Abstract Effective long term stable anaesthesia is a goal of many drug regimens employed in neuroscience in which procedures carried out are not practical in awake animals. A particular problem is the study of nociceptive mechanisms where good anaesthesia is essential. Similarly studies of cardiovascular or cerebrovascular mechanisms require that normal physiological reexes be preserved as much as is practical. For non-recovery anaesthesia h-chloralose is a good choice since it provides good anaesthesia without excess depression of physiological reexes. However, h-chloralose is sparingly soluble so that its use is not straightforward. We describe the characterisation of a simple procedure to solubilise h-chloralose in a solution of 2-hydroxypropyl-icyclodextrin. The resulting solution is stable at room temperature and gives a high concentration of h-chloralose making it easier to administer regularly during longer time course experiments. 1997 Elsevier Science B.V. Keywords: Anaesthesia; Cat; Fos; Pain

1. Introduction Long term stable effective anaesthesia is the goal of any regimen employed in neuroscience to allow studies that would not be practical using awake animals. Studies of pain pathways and nociceptive systems in particular, often require long term anaesthetic administration. For such studies h-chloralose has many biological advantages, particularly in not depressing cardiovascular and cerebrovascular reexes. However, its physiochemical properties are less than ideal and include poor solubility and a tendency to hydrolyse in solution at elevated temperatures. It is therefore of interest to examine ways in which the drug can be administered more efciently in longer term experiments. The choice of a general anaesthetic is discussed well
* Corresponding author. Tel.: + 44 171 8373611; fax: + 44 171 8130349; e-mail: peterg@brain.ion.ucl.ac.uk 0165-0270/97/$17.00 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 1 1 0 - 6

elsewhere (Flecknell, 1989). In essence, effective analgesia and anaesthesia are required for surgery, and gaseous anaesthetics are highly suited to that task. After surgical preparation an electrophysiological study may then make observations over many hours or a study, such as those involving Fos (Hunt et al., 1987), may require a long period of controlled observation. Such investigations are minimally invasive after surgery is complete and require long-lasting stable but not deep surgical anaesthesia. Similarly, studies in which cardiovascular or cerebrovascular responses are to be examined are of no value if appropriate physiological reexes are not active. In this regard, h-chloralose is an ideal agent in animal experiments because of the minimal depression of autonomic functions (Balis and Monroe, 1964; Bonvento et al., 1994). Preparations of chloralose (1,2-O-(2,2,2-trichloroethylidene)-h-D-glucofuranose) (White and Hixon, 1933) usually consist of a mixture of two iso-

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mers called h and i (Forsen et al., 1965) which have different physicochemical and pharmacological properties. The anaesthetic action of chloralose may be initially accompanied by spontaneous myoclonic movements and generalised clonic convulsions following abrupt peripheral stimulation (chloralose hyperexcitability). As a result of the mixed convulsant and anaesthetic properties of chloralose many conicting reports have appeared regarding its action (Balis and Monroe, 1964). As an explanation of its mixed action, it is commonly believed as proposed by some investigators (Chevalier and Cherbuliez, 1924; Kruger and Albe-Fessard, 1960) that h-chloralose has anaesthetic properties whereas i-chloralose causes the convulsant activity and is toxic. More recent studies (Monroe et al., 1963; Winters and Spooner, 1966) have found that the h-isomer has both properties and was 25 times more potent than the i-isomer. These ndings were contrary to those that previously reported that i-chloralose is toxic, since no deaths were observed even at the highest doses used (1600 mg/kg), as compared to the LD50 of h-chloralose at 120 mg/kg (in rats). It was also found that only occasional myoclonic movements were seen at extremely high doses. These important differences in the anaesthetic action of the two isomers are characteristic of other chloraloses (Butler, 1940). However, because of the high doses required to produce the effects with i-chloralose, the i-isomer may be a pharmacologically inactive compound as originally reported by Hanriot and Richet (1897) and the observed effects may have been due to contamination by h-chloralose. The potency data suggest a contamination of 4% h-chloralose in this case. Chloralose has poor solubility in water which places signicant limitations on its use. In water the i-isomer is much less soluble than the h-isomer (Windholz, 1976) allowing their separation by fractional recrystallization. The solubility of chloralose can be increased by heating. Chloralose is usually prepared as a 1% solution immediately before use by heating to 90C and then used at 40C before the h-chloralose has time to precipitate out of solution. It was recommended that the solution be ltered at 50C to remove i-chloralose (Dawson, 1969; Green, 1982) which precipitates rst because of its poorer solubility. This has been done because of the supposed toxic and convulsant properties of i-chloralose. However, in practical terms i-chloralose may lead to precipitation problems on administration. Earlier commercial preparations were highly contaminated with i-chloralose, however a recent preparation from Sigma contains only 5% of the i-isomer. Heating solutions of h-chloralose also causes signicant hydrolysis (Dastugue et al., 1955) and there are anecdotal suggestions that i-chloralose is formed. If further doses are to be given during the course of the experi-

ment the solution must be freshly prepared for each administration. In order to make the use of chloralose more convenient, solutions of 50 mg/ml chloralose in 25% urethane have been prepared. Urethane increases the aqueous solubility of chloralose (Green, 1982; Strobel and Wollman, 1969). However, due to the risks associated with urethane, it is hepatotoxic, suppresses haemopoiesis and acts as a carcinogen (Strobel and Wollman, 1969), its use is potentially hazardous. Alternately, chloralose may be prepared as a 1% solution in a warm (60C) solution of 10% aqueous polyethylene glycol and it will remain in solution on cooling (Green, 1982). The polyethylene glycol method still involves heating the chloralose and supports only dilute solutions. This paper describes a simple method for solubilising h-chloralose at relatively high concentrations using a commercially available i-cyclodextrin derivative that forms a soluble complex with the h-chloralose at room temperature. We have developed experimental models of head pain that require stable long term (2436 h) anaesthesia for both electrophysiological and Fos studies (Kaube et al., 1993). Our experience with the Fos technique, at least in the cat (Hoskin et al., 1996), is that a reasonable time is required between surgery and stimulation in the context of cranial nociception since the surgical eld is directly within the anatomical window of interest for the experiment. This paper describes a simple method for solubilising h-chloralose using a commercially available i-cyclodextrin derivative that forms a soluble complex with the hchloralose at room temperature. Furthermore, we have examined the effectiveness of this approach using Fos immunocytochemistry to ensure the solubilisation procedure did not adversely effect the anaesthetic potency of the drug.

2. Methods

2.1. h-Chloralosecyclodextrin complex

A solution of 2-hydroxypropyl-i-cyclodextrin (RBI, MA) was prepared as 4.5 g in 10 ml water for injection by stirring at room temperature. To this solution we added h-chloralose (Sigma, St Louis, MO, Cat N0 C9821, i-anomer:5%) at 50 mg/ml. After stirring at room temperature for 1 h any remaining undissolved chloralose and particulate matter was removed by ltration through a Millex-HA lter (0.45 vm; Millipore, MA). This gave a nal working concentration of h-chloralose suitable for intravenous administration at a dose of 0.4 ml/kg (20 mg/kg) for the maintenance of anaesthesia.

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2.2. Animal study

Cats were anaesthetised with halothane (1 3%) for surgery and h-chloralose for induction (60 mg/kg, intraperitoneally, as a solution in water as described in Section 1) and maintenance (15 20 mg/kg intravenous every 2 h as a cyclodextrin complex). The femoral artery and vein were cannulated in order to measure blood pressure and heart rate and provide access for drug and uid administration, respectively. The animals were endotracheally intubated, ventilated with 40% oxygen and paralysed after the surgical procedures with repeated doses of gallamine triethiodide (6 mg/kg intravenously; May and Baker, UK). Body temperature and end-expiratory CO2 were monitored and maintained within physiological limits. The animals were mounted in a stereotaxic frame and a circular midline craniotomy (2 cm in diameter) was performed for access to the superior sagittal sinus. The adjacent dura and falx were dissected parallel to the sinus over 10 15 mm. To prevent dehydration and for electrical insulation against the cortex a parafn bath was built with a dam of dental acrylic around the craniotomy and additionally a small polyethylene sheet inserted under the vessel. Fluid (4% glucose with 0.18% saline or normal saline) was administered intravenously at a rate of 3 5ml/kg per h.

(SIU5A, Grass Instruments, Quincy, MA; 150 V, 250 vs duration) at a rate of 0.3/s for 1 or 2 h.

2.4.1. Perfusion Cats were perfused transcardially with 11.5 l 0.9% saline after a bolus injection of 1000 I.U. heparin and 0.5 ml 1% sodium nitrite. This was followed by 2 l 4% paraformaldehyde in phosphate buffer (pH 7.4) and nally by 500600 ml 30% sucrose solution in phosphate buffer. The brain and cervical spinal cord were removed and stored in 50% sucrose with azide. Coronal sections (40 vm) of the caudal medulla and upper cervical spinal cord were cut on a freezing microtome and every fth section was collected for processing. Sections were cut from a block beginning at the level of the obex through to the C3 segment of the cervical cord. 2.4.2. Fos procedure Free-oating sections were incubated at 4C for 37 days in a rabbit, polyclonal anti-body to Fos protein (a generous gift of Dr Gerrard Evan) in a 1:1000 dilution with 1% phosphate buffered horse serum, containing 0.1% bovine serum albumin and 0.2% Triton-X100. Fos-like immunoreactivity (hereafter simply called Fos) was visualised using standard avidinbiotin peroxidase immunohistochemical techniques. Following the primary incubation, sections were washed in 0.1 M phosphate buffer (pH 7.4) for 30 min and then incubated in a biotinylated goat anti-rabbit IgG (1:200 dilution; Vector Labs, USA) for a minimum of 2 h at room temperature on a rotating table. Following the second incubation, the sections were washed again in 0.1 M phosphate buffer (pH 7.4) for 30 min. The sections were then incubated for 2.5 h in a 1:1000 dilution of ExtrAvidinperoxidase (Sigma, St. Louis, MO), sections were again washed in 0.1 M phosphate buffer (pH 7.4) for 30 min. The sections were then incubated in 20 ml 0.1 M phosphate buffer containing 0.05% diaminobenzidine (Sigma), 0.005% 4% ammonium chloride, 0.005% 20% D-glucose and 0.02% 1% solution of nickel ammonium sulphate for 20 min. The sections were then placed in a fresh identical 20 ml solution and 20 vl glucose oxidase (Sigma) was added to initiate the chromogenic reaction. The reaction was allowed to proceed until Fos positive nuclei could be clearly seen under the microscope. The DAB reaction product was visible as a black precipitate due to the presence of nickel ammonium sulphate. Following this reaction the sections were washed 23 times in 0.1 M phosphate buffer (pH 7.4) to terminate the reaction and then mounted. Fos-positive cells were distinguished by their black nickel enhanced nuclei from the background. Using the procedure adopted by Hammond et al. (1992), cells were only considered positive if the black precipitate of the DAB reaction within the cell nucleus was distin-

2.3. Assessment of anaesthesia

Adequacy of anaesthesia was monitored by observing cardiovascular and pupillary changes in response to pinch of the forepaw and the withdrawal response checked prior to re-administration of gallamine. Blood pressure and heart rate were stable and within physiological range for all animals throughout the whole experiment. Arterial blood gas parameters were monitored intermittently as a guide to the end-expiratory CO2 output.

2.4. Experiment design and stimulation

Following completion of surgery the animal was maintained essentially undisturbed for the following 24 h. The animals were then randomised to have either electrical stimulation or sham stimulation (electrode placement without activation). For electrical stimulation the superior sagittal sinus was suspended over a pair of platinum hook electrodes. We have previously shown that this would, of itself, not provoke signicant Fos activation (Kaube et al., 1993). We have compared here the level of Fos activation seen in control studies that used heated and non-heated (i-cyclodextrin) approaches to h-chloralose and similarly activation studies. The superior sagittal sinus was stimulated with a Grass S88 stimulator driving a stimulus isolation unit

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guishable from the background throughout a range of magnications between 20 to 4. Fos-positive cells were plotted onto sections of the caudal medulla and upper cervical spinal cord modied from published atlases (Berman, 1968; Rexed, 1954).

2.4.3. Plotting and statistics Distributions of cells were quantied for each individual animal by taking 10 sections at random, from each of the levels (trigeminal nucleus caudalis, C1, C2 and C3) and plotting the label from a single side onto one of the schematic sections described above. The plotting was performed by one person who, although they had knowledge of the experimental design, was not aware of the experimental group to which each animal belonged. The data are reported as a median with interquartile (25, 75%) ranges and have been compared using a Mann-Whitney U test in view of the fact that the Fos method as applied here can only yield non-continuous rather than interval data (Siegel, 1956).

median control levels of 7, 5 and 2 cells positive, respectively in laminae I/IIo of the trigeminal nucleus caudalis (TNC) and C1 and C2 cervical spinal cord. Electrical stimulation of the superior sagittal sinus (n= 4) produced reproducible clear increases in Fos expression in laminae I/IIo of the trigeminal nucleus caudalis and C1 and C2 cervical spinal cord. Stimulation produced a median of 81 (range 76114), 88 (range 84 107) and 92 (range 7097) Fos-positive cells, respectively in these regions. The increases seen were statistically signicant (PB0.05).

4. Discussion These data demonstrate the utility of a simple approach to solubilising a biologically useful compound, h-chloralose. We have shown that the aqueous solubility of the h-chloralose is markedly enhanced by dissolving it in a solution of 2-hydroxypropyl-i-cyclodextrin. The level of anaesthesia was unaffected by the solubilisation process as all physiological markers were unaltered. By employing the Fos methodology we have shown that the increased solubility of the h-chloralose complex provides no penalty in terms of increased Fos expression in control animals nor does it affect reactivity to stimuli. Cyclodextrins are cyclic oligosaccharides comprised of six (h), seven (i) or eight (k) glycopyranose units. They may be formed by the enzymatic degradation of amylose by cyclodextrin glycosyltransferases (EC 2.4.1.19) which results in a cleavage of the helix and reconnection into a circle at the h(14) bond. These molecules have a hydrophobic central cavity from which, and into which, non-polar molecules may enter and move away, a phenomenon known as inclusion or hostguest complexation (Pitha, 1989). The exterior of the molecules are sufciently hydrophilic to be soluble. Improvement in their solubility has been achieved by a condensation of the cyclodextrins with propylene oxide which yields 2-hydroxypropyl-cyclodextrins (Pitha et al., 1986). The selection of which cyclodextrin to use is determined by the chemical that one wishes to dissolve. It is related to a large degree to the hydrophobic cavity which varies from 5.2 A for the h form through 6.4 A for the i form to 8.3 A for the k form. If the guest molecule is too large the solution is not soluble and if it is too small the hostguest complexation is unstable. We have found by experience that 2-hydroxypropyl-icyclodextrin performs the task whereas h- and k-forms are unsuitable. In the studies we have performed we have seen no suggestion of toxicity or adverse events accompanying the administration of the 2-hydroxypropyl-i-cyclodextrinh-chloralose complex. This concurs with studies of h-, i- or k-cyclodextrin administration long term to

3. Results All animals included in the analysis were successfully maintained within normal physiological limits for the anaesthetised cat. There was no difference between the physiological data in either of the groups reported.

3.1. Stability of solution

h-Chloralose has excellent solubility in 45% (w/v) 2-hydroxypropyl-i-cylcodextrin solutions. The 2-hydroxypropyl-i-cyclodextrin h-chloralose complex solution was stable and we saw no evidence of re-crystallisation at room temperature or with storage at 4C.

3.2. Anaesthesia
Using the criteria as outlined in Section 2 experienced physiologists noted no difference in the level of anaesthesia in the cats receiving h-chloralose in cyclodextrin. Their cardiovascular (blood pressure and heart rate) responses were inhibited to noxious stimulation. When unparalysed there was no withdrawal response and no pupillary responses were observed during strong stimulation of the forepaw. We concluded from our routine monitoring of the animals that the effectiveness of h-chloralose was unaffected by solubilisation with cyclodextrin.

3.3. Fos studies

Seven cats were studied. Sham stimulation (placement of the electrode but no current, n = 3) produced

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mice in which no toxic effects were demonstrated (Pitha and Pitha, 1985). Our data suggest that solubilising h-chloralose with 2-hydroxypropyl-i-cyclodextrin does not reduce the anaesthetic potency. The h-chloralose remains in the complexed state as it is effectively driven into the guesthost complex by hydrophobic interactions in solution. Furthermore, the high concentration of the cyclodextrin favours re-formation of the complex should dissociation take place. After dilution in the plasma with administration the drug is able to dissociate from the complex by leaving the hydrophobic cavity and can be absorbed by the subjects tissues. This work describes a simple approach to a problem that is widespread in neuroscience. We have seen in other experiments using laser Doppler owmetry to measure local cerebral blood ow that vascular reactivity has been maintained, and in electrophysiological studies that trigeminal evoked potential activity is unaffected, by administration of h-chloralose as complexed with 2-hydroxy-i-cyclodextrin. Often neuroscientists would wish to use drugs, such as the anaesthetic h-chloralose, but their poor solubility mitigates against their use. Given the ease of use of this preparation of h-chloralose we would suggest that others may wish to evaluate this mode of solubilising h-chloralose in other experimental settings. Complexation of h-chloralose with 2-hydroxypropyl-i-cyclodextrin results in a solution that is easy to use and store, and an anaesthesia that is both robust and relatively free from unwanted, electrophysiological, cerebrovascular or cardiovascular side effects.

Acknowledgements The authors thank Dr Gerrard Evan, Imperial Cancer Research Fund, London, for the generous gift of the Fos antibody. We also thank Ms Y. Knight, Mr P. Hammond and Mr M. Lasalandra for expert technical assistance. This work has been supported by the Wellcome Trust and the Migraine Trust. PJG is a Wellcome Senior Research Fellow.

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