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Rapid identification of correctly located intracerebroventricular cannula in the rat
Journal of Neuroscience Methods, 8 (1983) 381-384 Elsevier
381
Rapid identification of correctly located intracerebroventricular cannula in the rat Melvyn P. Heyes, Stephen G a r n e t t and G e o f f e r y Coates Department of Nuclear Medicine, McMaster University Medical Centre, Hamilton, Ont. LSN 3Z5 (Canada) (Received March 7th, 1983) (Revised April 23rd, 1983) (Accepted May 4th, 1983)
Key words: intracerebroventricular injections--radiocontrast media--neurotoxins We describe a radiographic technique to identify a correctly located cannula in the ventricular system of the rat brain during life. A suspension of thorium dioxide was infused into the left lateral ventricle through a cannula placed in the brain under stereotaxic guidance. Correctly located injections were identified by the presence of a distinct thorium shadow on lateral X-ray of the in situ brain. When 6-hydroxydopamine was co-injected with thorium the resultant depletion of striatal dopamine and the changes in a conditioned avoidance responses were correlated with the degree of spreading of thorium through the ventricular system.
Introduction
Neurotoxins and neurotransmitter receptor ligands are frequently infused into the intracerebroventricular system of experimental animals when they cannot easily cross the blood-brain barrier or when peripheral effects are to be minimized. (Laverty and Taylor, 1970; Breese and Traylor, 1971; Lin et al., 1980; Avery et al., 1981; Willoughby and Day, 1981). The site of injection is usually confirmed by histological examination of post-mortem tissue. In studies where biochemical measurements are made it is difficult to exclude animals where the cannulae are incorrectly located. In this paper we describe a simple radiological technique which allows identification during life of those brains into which correctly located injections have been made.
Materials and Methods
Adult male Sprague-Dawley rats weighing 275-400 g at the time of the study were anaesthetized with sodium pentobarbital (50 mg/kg) and immobilized in a stereotaxic frame. After drilling a 2 mm diameter hole through the skull, 20 ~1 of a 25% colloidal suspension of thorium dioxide (Thorotrast: Fellows Testagar, Anaheim, 0165-0270/83/$03.00 © 1983 Elsevier Science Publishers B.V.
382 CA, U.S.A.) was infused into the left lateral ventricle at 1.5 /~l/min. Coordinates were derived from the n e u r o a n a t o m i c a l atlas of Pellegrino and C u s h m a n (1967) using bregma as zero point ( L = 1.6 mm, A - P = 3 . 2 to 3.6 ram, D V = 0 . 0 mm). Three minutes after the infusion had been completed the c a n n u l a was slowly removed and the hole filled with bone wax. Anaesthetized animals were X-rayed using a Siemen's G i g a n t o s X-ray generator with a 0.2 m m focal spot at a magnification of × 3. Kodak X - O m a t was used with an ultradeltoid screen. Frozen sections of excised brains were examined histologically. We measured the effect of an intracerebroventricular injection of the neurotoxin 6 - h y d r o x y d o p a m i n e dissolved in thorium dioxide ( 6 - O H D A , 20 p.g; n = 6) on the c o n c e n t r a t i o n s of dopamine, dihydroxyphenylacetic acid ( D O P A C ) and norepineph-
Fig. 1. Lateral X-ray view of in situ brain with an intracerebroventricular injection of 20 ~l of thorium dioxide. The thorium shadow is enclosed in the box.
383 rine in the striatum and hypothalamus. A group of animals which received only thorium dioxide (Sham; n = 5) and a group of unoperated animals (Intact; n = 5) acted as controls. In 6-OHDA animals we also measured the effects of catecholamine depletion on a conditioned avoidance response in which animals ran on a motor driven treadmill to avoid an electric grid placed at the rear of the belt. The animals ability to run was scored on a scale of 0 to 4.
Results The presence of a distinct thorium shadow on the lateral X-ray view of the in situ brain was unequivocably correlated with the penetration of the lateral ventricle by the cannula as confirmed by histological examination. The shape of the thorium shadow matched that of the ventricular system. It was generally elongated with distinct 'horns' (Fig. 1). No shadows were observed in A - P view. An injection of thorium dioxide alone into the lateral cerebral ventricles was without effect on the concentration of dopamine, D O P A C and norepinephrine in the striatum and hypothalamus (Sham vs Intact). In contrast, an injection of 6-OHDA into the lateral cerebral ventricle resulted in a significant depletion of norepinephrine in the striatum ( - 6 0 % , P < 0.01) and hypothalamus ( - 8 0 % , P < 0.02) and a significant depletion of dopamine ( - 4 0 % , P < 0.05) and DOPAC ( - 50%, P < 0.01) in the striatum, but no changes in the hypothalamus, compared to Sham. There was a significant correlation between the concentrations of dopamine and DOPAC in the striatum in 6-OHDA treated rats (r = 0.93, P < 0.01). When the degree of spreading of the thorium shadow was quantified by a score from 0 to 5 there was a significant correlation between the X-ray score and the concentration of dopamine (r = - 0 . 8 8 . P < 0.05) and the concentration of DOPAC (r = -0.90, P < 0.01) in the striatum. Catecholamine depleted animals also had impairments in the performance of the conditioned avoidance response, whereas in Sham animals their scores remained at pre-operative values. In 6-OHDA animals there was a significant correlation between the score of the conditioned avoidance response and the concentration of dopamine (r = 0.81, P < 0.05) and the concentration of DOPAC (r = 0.98, P < 0.001 ) in the striatum. There was also a significant correlation between the score of the conditioned avoidance response and the X-ray score (r = 0.89, P < 0.05).
Discussion This simple X-ray technique allows rapid identification of animals correctly injected into the lateral ventricle of the brain. The technique offers the advantage that animals with incorrectly located injections can be excluded from further biochemial and pharmacological analysis or from studies requiring chronic intracerebroventriclar cannulation. We found that in animals where injections were made directly into brain tissue a small shadow was visible in lateral view, presumably due to pooling of thorium in
384 the c a n n u l a track. It may be possible therefore to confirm the site of such injections on the basis of two X-ray views, one lateral the other d o r s a l - v e n t r a l . However, we usually find it difficult to visualize shadows in any other view than the lateral. Although other radiocontrast media may be used, t h o r i u m has several advantages. T h o r i u m has 4.9 times the X-ray stopping power as iodine (Johns and C u n n i n g h a m , 1971). T h o r i u m dioxide suspensions exert very little osmotic effect a n d do not cause i n f l a m a t i o n of the ependyma. These properties improve post-operative survival. Of 55 animals so injected, all but one survived. T h o r i u m dioxide also appears to be inert at least with respect to the concentrations of catecholamines in the brain and did not prevent the toxic effect of 6 - O H D A . The technique was also able to predict the degree of the biochemical and behavioural effects of 6 - O H D A .
Acknowledgements The technical assistance of Janice Skafel, George Joseph a n d Asghar Bhattis, D e p a r t m e n t of Radiology, is gratefully acknowledged.
References Breese, G.R. and Traylor, T.D. (1971) Depletion of brain noradrenaline and dopamine by 6-hydroxydopamine, Brit. J. Pharmacol., 42: 88-99. Hawkins, M.F. and Wunder, B.A. (1981) The effects of injections of bombesin into the cerebral ventricles on behavioural thermoregulation, Neuropharmacology, 20: 23-27. Johns, H.E. and Cunningham, J.R. (1971) The physics of Radiology (3rd edn.), Thomas, Springfield, IL, 152, pp. Laverty, R. and Taylor, K.M. (1970) Effects of intraventricular 2,4,5-tridhydroxyphenylethylamine(6-hydroxydopamine)on rat behaviour and brain catecholamine metabolism, Brit. J. Pharmacol., 40: 836-846. Lin, M.T., Chandra, A., Chern, Y.F. and Tsay, B.L. (1980) Intracerebroventricular injections of sympathomimetic drugs inhibit both heat production and heat loss mechanism in the rat, Canad. J. Physiol. Pharmacol 58: 896-902. Pellegrino, L.J. and Cushman, A.J. (1967) A Stereotaxic Atlas of the Rat Brain, Meridith, New York, NY. Willoughby, J.O. and Day, T.A. (1981) Central catecholamine depletion: effects on physiologicalgrowth hormone and prolactin secretion, Neuroendocrinology, 32: 65-69.
381
Rapid identification of correctly located intracerebroventricular cannula in the rat Melvyn P. Heyes, Stephen G a r n e t t and G e o f f e r y Coates Department of Nuclear Medicine, McMaster University Medical Centre, Hamilton, Ont. LSN 3Z5 (Canada) (Received March 7th, 1983) (Revised April 23rd, 1983) (Accepted May 4th, 1983)
Key words: intracerebroventricular injections--radiocontrast media--neurotoxins We describe a radiographic technique to identify a correctly located cannula in the ventricular system of the rat brain during life. A suspension of thorium dioxide was infused into the left lateral ventricle through a cannula placed in the brain under stereotaxic guidance. Correctly located injections were identified by the presence of a distinct thorium shadow on lateral X-ray of the in situ brain. When 6-hydroxydopamine was co-injected with thorium the resultant depletion of striatal dopamine and the changes in a conditioned avoidance responses were correlated with the degree of spreading of thorium through the ventricular system.
Introduction
Neurotoxins and neurotransmitter receptor ligands are frequently infused into the intracerebroventricular system of experimental animals when they cannot easily cross the blood-brain barrier or when peripheral effects are to be minimized. (Laverty and Taylor, 1970; Breese and Traylor, 1971; Lin et al., 1980; Avery et al., 1981; Willoughby and Day, 1981). The site of injection is usually confirmed by histological examination of post-mortem tissue. In studies where biochemical measurements are made it is difficult to exclude animals where the cannulae are incorrectly located. In this paper we describe a simple radiological technique which allows identification during life of those brains into which correctly located injections have been made.
Materials and Methods
Adult male Sprague-Dawley rats weighing 275-400 g at the time of the study were anaesthetized with sodium pentobarbital (50 mg/kg) and immobilized in a stereotaxic frame. After drilling a 2 mm diameter hole through the skull, 20 ~1 of a 25% colloidal suspension of thorium dioxide (Thorotrast: Fellows Testagar, Anaheim, 0165-0270/83/$03.00 © 1983 Elsevier Science Publishers B.V.
382 CA, U.S.A.) was infused into the left lateral ventricle at 1.5 /~l/min. Coordinates were derived from the n e u r o a n a t o m i c a l atlas of Pellegrino and C u s h m a n (1967) using bregma as zero point ( L = 1.6 mm, A - P = 3 . 2 to 3.6 ram, D V = 0 . 0 mm). Three minutes after the infusion had been completed the c a n n u l a was slowly removed and the hole filled with bone wax. Anaesthetized animals were X-rayed using a Siemen's G i g a n t o s X-ray generator with a 0.2 m m focal spot at a magnification of × 3. Kodak X - O m a t was used with an ultradeltoid screen. Frozen sections of excised brains were examined histologically. We measured the effect of an intracerebroventricular injection of the neurotoxin 6 - h y d r o x y d o p a m i n e dissolved in thorium dioxide ( 6 - O H D A , 20 p.g; n = 6) on the c o n c e n t r a t i o n s of dopamine, dihydroxyphenylacetic acid ( D O P A C ) and norepineph-
Fig. 1. Lateral X-ray view of in situ brain with an intracerebroventricular injection of 20 ~l of thorium dioxide. The thorium shadow is enclosed in the box.
383 rine in the striatum and hypothalamus. A group of animals which received only thorium dioxide (Sham; n = 5) and a group of unoperated animals (Intact; n = 5) acted as controls. In 6-OHDA animals we also measured the effects of catecholamine depletion on a conditioned avoidance response in which animals ran on a motor driven treadmill to avoid an electric grid placed at the rear of the belt. The animals ability to run was scored on a scale of 0 to 4.
Results The presence of a distinct thorium shadow on the lateral X-ray view of the in situ brain was unequivocably correlated with the penetration of the lateral ventricle by the cannula as confirmed by histological examination. The shape of the thorium shadow matched that of the ventricular system. It was generally elongated with distinct 'horns' (Fig. 1). No shadows were observed in A - P view. An injection of thorium dioxide alone into the lateral cerebral ventricles was without effect on the concentration of dopamine, D O P A C and norepinephrine in the striatum and hypothalamus (Sham vs Intact). In contrast, an injection of 6-OHDA into the lateral cerebral ventricle resulted in a significant depletion of norepinephrine in the striatum ( - 6 0 % , P < 0.01) and hypothalamus ( - 8 0 % , P < 0.02) and a significant depletion of dopamine ( - 4 0 % , P < 0.05) and DOPAC ( - 50%, P < 0.01) in the striatum, but no changes in the hypothalamus, compared to Sham. There was a significant correlation between the concentrations of dopamine and DOPAC in the striatum in 6-OHDA treated rats (r = 0.93, P < 0.01). When the degree of spreading of the thorium shadow was quantified by a score from 0 to 5 there was a significant correlation between the X-ray score and the concentration of dopamine (r = - 0 . 8 8 . P < 0.05) and the concentration of DOPAC (r = -0.90, P < 0.01) in the striatum. Catecholamine depleted animals also had impairments in the performance of the conditioned avoidance response, whereas in Sham animals their scores remained at pre-operative values. In 6-OHDA animals there was a significant correlation between the score of the conditioned avoidance response and the concentration of dopamine (r = 0.81, P < 0.05) and the concentration of DOPAC (r = 0.98, P < 0.001 ) in the striatum. There was also a significant correlation between the score of the conditioned avoidance response and the X-ray score (r = 0.89, P < 0.05).
Discussion This simple X-ray technique allows rapid identification of animals correctly injected into the lateral ventricle of the brain. The technique offers the advantage that animals with incorrectly located injections can be excluded from further biochemial and pharmacological analysis or from studies requiring chronic intracerebroventriclar cannulation. We found that in animals where injections were made directly into brain tissue a small shadow was visible in lateral view, presumably due to pooling of thorium in
384 the c a n n u l a track. It may be possible therefore to confirm the site of such injections on the basis of two X-ray views, one lateral the other d o r s a l - v e n t r a l . However, we usually find it difficult to visualize shadows in any other view than the lateral. Although other radiocontrast media may be used, t h o r i u m has several advantages. T h o r i u m has 4.9 times the X-ray stopping power as iodine (Johns and C u n n i n g h a m , 1971). T h o r i u m dioxide suspensions exert very little osmotic effect a n d do not cause i n f l a m a t i o n of the ependyma. These properties improve post-operative survival. Of 55 animals so injected, all but one survived. T h o r i u m dioxide also appears to be inert at least with respect to the concentrations of catecholamines in the brain and did not prevent the toxic effect of 6 - O H D A . The technique was also able to predict the degree of the biochemical and behavioural effects of 6 - O H D A .
Acknowledgements The technical assistance of Janice Skafel, George Joseph a n d Asghar Bhattis, D e p a r t m e n t of Radiology, is gratefully acknowledged.
References Breese, G.R. and Traylor, T.D. (1971) Depletion of brain noradrenaline and dopamine by 6-hydroxydopamine, Brit. J. Pharmacol., 42: 88-99. Hawkins, M.F. and Wunder, B.A. (1981) The effects of injections of bombesin into the cerebral ventricles on behavioural thermoregulation, Neuropharmacology, 20: 23-27. Johns, H.E. and Cunningham, J.R. (1971) The physics of Radiology (3rd edn.), Thomas, Springfield, IL, 152, pp. Laverty, R. and Taylor, K.M. (1970) Effects of intraventricular 2,4,5-tridhydroxyphenylethylamine(6-hydroxydopamine)on rat behaviour and brain catecholamine metabolism, Brit. J. Pharmacol., 40: 836-846. Lin, M.T., Chandra, A., Chern, Y.F. and Tsay, B.L. (1980) Intracerebroventricular injections of sympathomimetic drugs inhibit both heat production and heat loss mechanism in the rat, Canad. J. Physiol. Pharmacol 58: 896-902. Pellegrino, L.J. and Cushman, A.J. (1967) A Stereotaxic Atlas of the Rat Brain, Meridith, New York, NY. Willoughby, J.O. and Day, T.A. (1981) Central catecholamine depletion: effects on physiologicalgrowth hormone and prolactin secretion, Neuroendocrinology, 32: 65-69.