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CHRONIC NICOTINE PRETREATMENT PROTECTS THE BLOOD–BRAIN BARRIER AGAINST NICOTINE-INDUCED SEIZURES IN THE RAT
Pharmacological Research, Vol. 40, No. 3, 1999 Article No. phrs.1999.0514, available online at http:rrwww.idealibrary.com on
CHRONIC NICOTINE PRETREATMENT PROTECTS THE BLOOD – BRAIN BARRIER AGAINST NICOTINE-INDUCED SEIZURES IN THE RAT U ¨ ¨ ¨ ˙ ˙ GULAY UZUM, A. SARPER DILER and Y. ZIYA ZIYLAN
¸apa, 34390 Department of Physiology and Medical Biology, Istanbul Faculty of Medicine, C Istanbul, Turkey Accepted 18 March 1999
This study was designed to investigate the possible protective actions of nicotine on cerebrovascular permeability in convulsions during nicotine-induced seizures. We have measured the permeability changes in the blood]brain barrier ŽBBB. macroscopically and spectrophotometrically by using Evans blue dye. Specific gravity measurements were also performed to assess brain edema which develops after blood]brain barrier opening. The experiments were carried out on Wistar rats. Rats were divided into two groups. They received acutely a convulsive dose of nicotine 3, 5, 8 and 9 mg kgy1 i.p. or pretreated with a low dose of nicotine Ž0.8 mg kgy1 i.p.. for 21 days followed by the procedure mentioned in the first group. Acute nicotine injection induced a significant increase in blood pressure and Evans-blue passage, despite a decline in specific gravity values. Low doses of chronic nicotine administration markedly reduced both the leakage of dye, and brain water content. Chronic treatment with low doses of nicotine Ž0.8 mg kgy1 dayy1 s.c.. lessened the intensity of tonic]clonic seizures observed with a single dose of 3, 5, 8 or 9 mg kgy1 nicotine. The data presented here demonstrate that nicotine pretreatment results in decreased sensitivity to nicotine-induced seizures in rats. Q 1999 Academic Press KEY
WORDS:
nicotine, seizures, blood]brain barrier.
INTRODUCTION Nicotine, a tertiary amine composed of a pyridine and pyrolidine ring, is one of the most widely used substance by humans and has various complex actions. Owing to its lipid solubility it can readily pass into brain parenchyma crossing the blood]brain barrier ŽBBB.. This explains nicotine’s direct effect in the brain, one of which is neuroexcitation. It also operates specific receptors at neuromuscular junctions and in the brain biphasically and has dose-dependent mixed pharmacological effects w1, 2x. It has been shown that this biphasic action is composed of an initial transient stimulation followed by a long lasting depression known as desensitization. Recent in ¨ itro and in ¨ i¨ o investigations have been reported for the neuroprotective effects of nicotine against some excitotoxins w3]5x. High doses of nicotine injection has been also shown to cause hypertension and clonic]tonic convulsions w6, 7x U
Corresponding author.
1043]6618r99r090263]07r$30.00r0
which could both be directly related to microvascular permeability w8, 9x. Previous studies have suggested that nicotineinduced seizure sensitivity decreases as a consequence of nicotine pretreatment w10x. This study was designed to explore the possible beneficial effects of nicotine on vascular functions in the brain and motor convulsions associated with systemic nicotine injection. Vascular effects of nicotine were evaluated by measuring the changes of brain microvascular permeability, and specific gravity ŽSG. which indicates brain water and protein content. MATERIALS AND METHODS Adult male wistar rats ŽDETAM-TURKEY. weighing 200]250 g were used. Animals Ž n s 60. were randomly divided into two groups. One group was acutely treated with seizure-producing doses of nicotines w3, 5, 8 and 9 mg kgy1 i.p, N-3376 Žy. Nicotine free base SIGMAx. Another group of aniQ 1999 Academic Press
Pharmacological Research, Vol. 40, No. 3, 1999
264
mals was pretreated with a low dose of nicotine w11x Ž0.8 mg kgy1 dayy1 i.p.. for 21 days followed by the procedure mentioned in the first group. A control group of rats received either a single injection of the same amount of isotonic saline Ž0.9% NaCl., or daily injection for 21 days. Details of animal preparation and the measurements have been presented previously w2, 6x. Briefly, under light ether anaesthesia, catheters filled with heparinised saline were inserted into the femoral artery and vein and hind quarters of animals were immobilized in a loose-fitting plaster cast. Next the rats were then allowed to recover. The body temperature was monitored with a rectal probe, and external heating lamps were utilized to maintain body temperature at 35]378C. At least 30 min after anaesthesia, when the animals were entirely conscious, 3 ml kgy1 2% Evans blue ŽEB. solution was injected i.v. for visual examination of BBB distribution and spectrophotometric measurements. Ten minutes later seizure-producing doses of nicotine Ž3, 5, 8 and 9 mg kgy1 . were given. The behavioral characteristics of convulsions and duration of convulsions were observed in each individual animal for a period of 30 min. Mean arterial blood pressure was recorded continuously by connecting the arterial catheter to a polygraph system ŽNihon-Kohden, Tokyo, Japan.. At the end of the experiments, 60 min after the injection rats were killed and brains perfused transaortically with a saline solution and removed. After macroscopic evaluation of the brain surface, coronal sections were made and examined for the
extent and the intensity of staining due to EB extravasation. Barrier opening to intravascular EB albumin was graded from 0 Žno staining. to 3 q Ždeep blue. staining. Distribution of EB in different brain regions was also assessed quantitatively using the spectrophotometric method described by Rossner and Temple w12x. In brief, after perfused brains were divided into coronal sections, the wet tissue samples of the six different brain regions were weighed on preweighed aluminium foil, homogenized with 50% trichloroacetic acid in fivefold, centrifuged at 15,000 rpm for 20 min and absorbance was measured at 615 nm. Bilateral specimens of the same eight brain areas from the brain slices were dropped into a gradient column consisting of a heavy and light mixture of kerosene and monobromobenzene. The position which the tissue sample had reached in the column after 5 min was recorded and compared with that of standard K 2 SO4 solutions w13x. Electroencephalographic ŽEEG. recordings were performed in some rats to identify whether a clonic seizure took place and in each animal for the latency of the seizure. The rats in this series were tracheotomized and an artery and a vein were cannulated. The animals were then paralysed with Pavulon ŽOrganonrIstanbul, Turkey. and mechanically ventilated in an 70% N2 and 30% O 2 gas mixture. Xylocaine ŽEczacıbas¸ırIstanbulrTurkey . was employed to infiltrate all surgical wounds. Frontoparietal screw type electrodes were placed through the skull on each side in contact with the meninges.
Table I Mean arterial blood pressure (MABP), blood brain barrier leakage in convulsions induced by various doses of acute nicotine and the effect of chronic nicotine pretreatment Groups (n )
Mean arterial blood pressure (mmHg) Maximum Increase (D P)
Initial Control Ž5. Acute nicotine 3 mg kgy1 Ž5. 5 mg kgy1 Ž5. 8 mg kgy1 Ž5. 9 mg kgy1 Ž5. Choronic nicotine pretreatment q acute nicotine 3 mg kgy1 Ž5. 5 mg kgy1 Ž5. 8 mg kgy1 Ž5. 9 mg kgy1 Ž5. U
100 " 5
]
]
94 " 4 98 " 6 100 " 5 96 "
120 " 5 160 " 8U 165 " 9U 170 " 10U
26 62 65 74
118 " 5 125 " 6UU 123 " 6UU 123 " 6UU
130 " 6 165 " 7 169 " 7 172 " 6
12 UUU 40 UUU 46 UU 48
UUU
Duration (min) Dt
0
q0.5
q1
q2
q3
]
5
]
]
]
]
] ] ] ]
2 ] ] ]
3 3 1 1
] 2 4 3
] ] ] 1
5 4 4 3
] 1 1 2
] ] ] ]
] ] ] ]
] ] ] ]
7 "3 9 "4 10 " 3 12 " 5
2 "0.5UUU 3 "0.5UUU 3 "0.6UUU 4 "1UUU
Degrees of BBB breakdown
Comparison between control values, significance taken as P - 0.01; UU comparison between control onset values, significance taken as P- 0.05; UUU comparison of D P and D t values with acute nicotine groups, significance taken as P - 0.01. 0, no staining; q0.5, local very light blue staining; q1, local light blue staining; q2, generalized moderate staining; q3, generalized deep blue staining.
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Recordings were made on a polygraph system with an amplification sensitivity of 140 mV cmy1 and at a speed of 25 mm sy1 . Statistical analysis was performed using Student’s t-test.
RESULTS Table I lists the mean arterial blood pressure ŽMABP., maximal MABP and the D P in salinetreated controls and rats treated with nicotine. The mean systemic blood pressure in anaesthetized rats at onset was 100 " 5 mmHg in all rats apart from rats treated with low doses of nicotine. Blood pressure increased by approximately 60]70 mmHg and lasted for 7]10 min. The MABP in rats pretreated with low doses of nicotine was held firmly near 125 mmHg. Acute nicotine administration to this group increased MABP by 40]45 mmHg for 2]3 min. Therefore D P and D t were significantly lower than groups treated only with acute nicotine. With acute nicotine injection, spike wave activity and high-voltage potentials were seen in EEG recordings. This activity was maximum in the third
minute and gradually decreased by 10]12 min. The sequence of behavioural changes started with head twitching and clonic movements of forelimbs progressing to generalized tonic]clonic convulsions in 1]2 min Ži.e. latencies . after 3, 5, 8 and 9 mg kgy1 acute injection of nicotine. Low doses of nicotine-pretreated rats were less sensitive to the seizure-producing effect of nicotine than are saline-pretreated rats. Furthermore, rats that are pretreated with nicotine have relatively longer latencies to nicotine-induced seizures with respect to saline-treated controls. Latencies to seizures decreased with increased challenging doses of nicotine. Chronic treatment with low doses of nicotine prevented the occurrence of tonic clonic seizures observed with a single dose of 3, 5, 8 and 9 mg kgy1 nicotine and EEG recordings showed marked attenuation in the frequency and the intensity of spikewaves. In acute nicotine-injected rats, the BBB breakdown to EB was seen and was characteristically always confined to anatomically-limited areas. The regional distribution of BBB breakdown ŽFig. 1. and its intensity are summarized in Table I. With increased challenging doses of nicotine more intense
Fig. 1. legend over page.
266
Pharmacological Research, Vol. 40, No. 3, 1999
Fig. 1. Spectral analysis of Evans Blue dye in convulsions induced by various doses of acute nicotine and the effects of chronic nicotine pretreatment in cortex, hippocampus, midbrain, striatum, thalamus, cerebellum. mg kgy1A stands for acute nicotine groups, mg kgy1 stands for choronic pretreatment groups receiving acute nicotine. Comparison of chronic pretreatment groups receiving acute nicotine with acute nicotine groups U Significance taken as P - 0.05.
1.0420" 0.55 1.0415" 0.42 1.0424" 0.21 1.0425" 0.35 1.0422" 0.48 1.0450" 0.35 1.0420" 0.43
3 (n s 8)
1.0408" 0.31U 1.0401" 0.35U 1.0415" 0.34 1.0417" 0.34U 1.0423" 0.41 1.0442" 0.39 1.0410" 0.38U
5 (n s 8)
1.0390" 0.19 1.0385" 0.21U 1.0408" 0.19 1.0406" 0.27U 1.0419" 0.29 1.0430" 0.31 1.0395" 0.26U
8 (n s 8)
Acute nicotine (mg kg y 1)
1.0390" 0.27U 1.0383" 0.25U 1.0400" 0.21 1.0405" 0.27U 1.0420" 0.37 1.0428" 0.39 1.0386" 0.41U
9 (n s 8)
1.0448" 0.43 1.0438" 0.33 1.0419" 0.31U 1.0386" 0.25U 1.0381" 0.44
1.0435" 0.49 1.0425" 0.42 1.0430" 0.51 1.0443" 0.45 1.0438" 0.41 1.0465" 0.38 1.0436" 0.38
Control (n s 8)
U
1.0446" 0.39
1.0425" 0.31 1.0425" 0.33 1.0429" 0.37 1.0444" 0.31 1.0438" 0.30 1.0464" 0.34 1.0429" 0.35
21 day chronic nicotine treatment (0.8 mg kg y 1 day y 1) (n s 8) 1.0425" 0.37UU 1.0413" 0.32 1.0428" 0.33 1.0440" 0.34UU 1.0430" 0.36 1.0445" 0.36 1.0430" 0.36UU
5 (n s 8)
1.0421" 0.29UU 1.0415" 0.36 1.0425" 0.33 1.0438" 0.34UU 1.0428" 0.37 1.0447" 0.36 1.0428" 0.34UU
8 (n s 8)
1.0413" 0.31UU 1.0413" 0.31 1.0420" 0.29 1.0437" 0.36UU 1.0428" 0.33 1.0445" 0.35 1.0425" 0.27
9 (n s 8)
1.0445" 0.33UU 1.0440" 0.40UU 1.0437" 0.38UU 1.0435" 0.36UU
1.0430" 0.31 1.0420" 0.34 1.0428" 0.36 1.0445" 0.36UU 1.0436" 0.37 1.0460" 0.38 1.0434" 0.32UU
3 (n s 8)
Acute nicotein (mg kg y 1)
Comparison of chronic pretreatment groups receiving acute nicotine with controls nicotine groups. Significance taken as P - 0.05. UU Comparison of chronic pretreatment groups receiving acute nicotine with acute nicotine groups Significance taken as P - 0.05.
Brain regions Hippocampus Midbrain Hypothalamus Thalamus Striatum Cerebellum Corpus collosum Cortex
Groups
Table II Specific Gravity measurements in convulsions induced by various doses of acute nicotine and the effects of chronic nicotine pretreatment
Pharmacological Research, Vol. 40, No. 3, 1999 267
268
BBB breakdown to EB was seen. A 3 q staining was present with 9 mg kgy1 acute nicotine. There was marked difference between BBB interrupting acute nicotine groups with corresponding chronic nicotine pretreatment groups Ž P- 0.051. although, chronic nicotine pretreatment in this study could not completely suppress EB extravasation ŽFig. 1.. SG measurements showed significant differences between control and acute nicotine groups. Corresponding dose groups of cerebellum, cortex, hippocampus Ži.e. Acute ¨ s pretreatment groups. showed significant variations Ž P- 0.05.. There was no significant SG value difference between controls and pretreated groups in midbrain, hypothalamus, caudate nucleus and cerebellum ŽTable II.. Low doses of chronic nicotine administration markedly reduced the leakage of EB and rendered increased SG values reaching to controls ŽFig. 1 and Table II.. Blood]brain barrier opening to EB was consistently present in those experimental groups with a MABP increase at seizure onset ŽTable I..
DISCUSSION
Considerable studies have shown that epileptic seizures may induce neuronal damage and that seizures are accompanied by an abrupt increase in blood pressure and this interacts directly with cerebral blood flow and microvascular permeability w14]17x. In our study, nicotine-induced generalized convulsions were accompanied by elevated MABP scores, in accordance with previous studies w7, 16, 18, 19x. As in many cases, a decreased specific gravity value and regional BBB breakdown Žespecially cortex, hippocampus. were also found w16x. An acute rise in MABP was somewhat smaller, and extravasation of EB was less extensive and SG values were increased in rats treated with a subseizure-producing dose of nicotine. In these rats initial MABP was higher than those of controls before challenging with high doses of nicotine. Since the degree of leakage in epileptic seizures are directly related to the magnitude of systemic blood pressure, it seems that nicotine treatment protected the BBB by limiting the severe elevation of D P. In addition, it is likely that nicotine may alter cerebral blood flow by changing the cerebral circulatory response w6x. Moreover, the administration of nicotine can modify the release of a number of both neurotransmitters and neurohormones w20x. Among the hormones, whose levels are modulated by nicotine, ACTH has been reported that it has to have a role in stimulating the release of steroids such as cortisol from the adrenal cortex w21x.
Pharmacological Research, Vol. 40, No. 3, 1999
A relatively new aspect of nicotine’s action has been put forward by Owman et al. w22x. In their studies they have employed nicotine pretreatment on rats whose meso-striatal dopamine system had been transected. They have found amelioration in blood flow and glucose utilization in caudate putamen on the side where meso-striatal dopamine system had been transected. These observations and our previous studies which demonstrated that the administration of glucocorticoids produced a decreased cerebrovascular permeability, while withdrawal of the drug resulted in increased permeability w23, 24x which makes it possible to speculate that the pituitary]adrenal axis might also play an important role in the regulation of permeability of brain microvasculature in nicotine-pretreated animals. It is well accepted that low doses of nicotine interacts biphasically with specific receptors in the brain. This biphasic action is similar to an initial transient stimulation followed by a more long lasting depression known as desensitization w10x. At higher doses nicotine has been shown to cause clonic]tonic convulsions w1, 7, 19x. De Fiebre et al. w10x showed that nicotine pretreatment rendered animals less susceptible to nicotine-induced seizures than salinepretreated controls. The data presented here clearly demonstrated that nicotine-pretreated animals are less sensitive to the seizure producing effects of nicotine than saline-pretreated rats. Furthermore, rats that are pretreated with nicotine have significantly longer latencies to nicotine-induced seizures. One possible explanation for both the decreased sensitivity and longer latencies is nicotinic-receptor desensitisation or inactivation in CNS caused by low-dose nicotine treatment. Indeed, Simoska et al. w25x have shown that desensitisation and inactivation occur in nicotinic receptors after treatment with nicotine agonists. In addition, decreased sensitivity to seizures reported here could also be caused by a number of other factors such as an active metabolite of nicotine Ži.e. nicotine N-oxide. by inhibiting nicotine’s action as suggested Barras et al. w26x. Finally this work showed that nicotine pretreatment may have protective actions on neuronal and vascular functions in the brain against nicotineinduced seizures, shown by normalized EEG activity in addition to increased SG values Ži.e. getting closer to control SG values. and decreased EB extravasation Žby spectrophotometry.. This protective effect could be explained by the nicotine-induced desensitization or inactivation of the nicotinic receptors in the CNS. The observed changes indicating the protective effects in the permeability of BBB and specific gravity, may be partially due to secondary metabolic activation of nicotine on the cerebral microenvironment.
Pharmacological Research, Vol. 40, No. 3, 1999
ACKNOWLEDGEMENTS This work was supported by the Research Fund of the University of Istanbul 660-210994.
REFERENCES 1. Court JA, Perry EK. Nicotine receptors. Pharmacol Pathophysiol 1994; 2(3): 216]33. 2. Wecker L, Jian Lu Z. CNS neurotransmitters and neuromodulators, acetylcholine. In: Function of nicotinic receptors in CNS. TW Stone ed. Boca Raton FL: CRC Press, 1995: 105]14. 3. Abood LG, Salles A, Maiti A. Structure]activity studies of carbamat and other esters: Agonists and antagonists to nicotine. Pharmacol Biochem Beha¨ 1987; 30: 403]8. 4. Borlongan CV, Shytle RD, Ross SD, Shimizu T, Frreman TB, Cahill DW, Sanberg PR. Žy. Nicotine protects against systemic kainic acid-induced exitotoxic effects. Exp Neurol 1995; 136: 261]5. 5. Marin P, Maus S, Desagher J. Gaownski A, Premont J. Nicotine: A new neuroprotective agent. Neuroreport 1994; 5: 1977]80. ¨ ¨ G, Lefauconnier JM, Ziylan YZ. 6. Diler AS, Uzum Biology and physiology of the blood]brain barrier. In: Ad¨ ances in beha¨ ioral biology. Couraud P-O, Scherman D, eds. New York: Plenum Press, 1996; 46: 333]8. ¨ ¨ G, Curgunlu S, Hacıalioglu 7. Uzum ˘ M, Ercan S, Diler AS, Ziylan YZ. Protective effect of chronic administration of nicotine on neuronal and vascular functions in the brain. Eur J Neurosci 1993; Suppl 6: 93S358. 8. Johonsson B, Li CH, Olsson Y, Klatzo J. The effect of acute arterial hypertension on the blood]brain barrier to protein tracers. Acta Neurol Pathol 1970; 16: 117]24. 9. Ziylan YZ, Astes N. Age-related changes in regional patterns of blood]brain barrier breakdown during epileptiform seizures induced by pentylenetetrazol. Neurosci Lett 1989; 96: 179]84. 10. De Fiebre C, Collins AC. Decreased sensitivity to nicotine induced seizures as a consequence of nicotine pretreatment with Long-Sleep and Short-Sleep Mice. Alcohol 1988; 5: 55]61. 11. Hulien-Gublin BA, Lumpkin MD, Kellar KJ. Effects of chronic administration of nicotine on prolactin release in the rat: Inactivation of prolactin response by repeated injections of nicotine. J Pharmacol Exp Ther 1990; 252: 21]5. 12. Rossner N, Tempel K. Quantitative Bestimmung der ¨ Permeabilitat ¨ der sagenonnten blut-hirnschranke fur ¨ Evans-Blau ŽT 1824.: Med Pharmacol Exp 1966; 14: 169]82. 13. Nelson S, Manz M. Use of specific gravity in the measurement of cerebral edema. J Appl Physiol 1971; 30: 268]71.
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14. Bolwing TG, Hertz MM, Holm JJ. Blood brain barrier permeability during electroshock seizures in the rat. Eur J Clin In¨ est 1977; 7: 95]100. 15. Lee JC, Olszewki J. Increased cerebrovascular permeability after repeated electroshock. Neurology 1961; 11: 515]19. 16. Nitsch C, Klatzo I. Regional patterns of blood]brain barrier breakdown during epilepti form seizures induced by various convulsive agents. J Neurol Sci 1983; 59: 305]22. 17. Ziylan YZ. Pathophysiology of the opening of the blood]brain barrier and blood-cerebro spinal fluid barriers in acute hypertension. Exp Neurol 1984: 18]28. 18. Nitsch C, Goping G, Klatso I. Pathophysiological aspects of blood]brain barrier permeability in epileptic seizures. In: Ad¨ ances in experimental medicine and biology, ¨ ol 203. Exitatory amino acids and epilepsia. Schwarz R, Ben-Ari Y, eds. New York, London 1986: 175]84. 19. Wecker L, Yu ZJ. Function of nicotinic receptor in the CNS. In: CNS Neurotransmitter and neuromodulators. Boca Raton, FL: Acetylcholine CRC Press, 1995: 105]14. 20. Summers KL, Glacabini E. Effects of local and repeated systemic administration of Žy. nicotine on extracellular levels of acetylcholine, norepinephrine, dopamine, and serotonin in rat cortex. Neurochem Res 1995; 20: 753]9. 21. Pomerleau OF, Rosecrans JI. Neuroregulatory effects of nicotine. Psychoneuroendocrinology 1989; 92: 135]43. 22. Owman C, Fuxe K, Janson AM, Kahstrom ¨ J. Studies of protective actions of nicotine on neuronal and vascular functions in the brain of rats; comparison between sympathetic noradrenaergic and mesostriatal dopaminergic fiber systems and the effect of a dopamine agonist. In: Progress in brain research. Nordberg A, Fuxe K, Holmsted B, Sudwall A, eds. Vol 79, Chap 26. Elsevier Science Publishers BV 1989: 267]76. 23. Ziylan YZ, Lefauconnier JM, Bernard G, Bourre JM. Regional alterations in blood to brain transfer of a-aminoisobutyric acid and sucrose after chronic administration and withdrawal of dexamethasone. J Neurochem 1989; 52: 684]90. 24. Ziylan YZ, Lefauronnier JM, Bernard G, Bourre JM. Hormonal influence on the permeability of the blood]brain barrier: effect of an analog of adrenocorticotropic hormone, b 1-24 corticotrophin. Neurosci Lett 1993; 151: 59]63. 25. Simasko SM, Soares JR, Weiland GA. Two components of carbomylcholine-induced loss of nicotinic acetylcholine receptor function in the neuronal cell line PC 12. Mol Pharmacol 1986; 30: 6]12. 26. Barrass BC, Blackburn JW, Brimblerombe RW, Rich P. Modifications of nicotine toxicity by treatment with different drugs. Biochem Pharmacol 1969; 18: 2145]52.
CHRONIC NICOTINE PRETREATMENT PROTECTS THE BLOOD – BRAIN BARRIER AGAINST NICOTINE-INDUCED SEIZURES IN THE RAT U ¨ ¨ ¨ ˙ ˙ GULAY UZUM, A. SARPER DILER and Y. ZIYA ZIYLAN
¸apa, 34390 Department of Physiology and Medical Biology, Istanbul Faculty of Medicine, C Istanbul, Turkey Accepted 18 March 1999
This study was designed to investigate the possible protective actions of nicotine on cerebrovascular permeability in convulsions during nicotine-induced seizures. We have measured the permeability changes in the blood]brain barrier ŽBBB. macroscopically and spectrophotometrically by using Evans blue dye. Specific gravity measurements were also performed to assess brain edema which develops after blood]brain barrier opening. The experiments were carried out on Wistar rats. Rats were divided into two groups. They received acutely a convulsive dose of nicotine 3, 5, 8 and 9 mg kgy1 i.p. or pretreated with a low dose of nicotine Ž0.8 mg kgy1 i.p.. for 21 days followed by the procedure mentioned in the first group. Acute nicotine injection induced a significant increase in blood pressure and Evans-blue passage, despite a decline in specific gravity values. Low doses of chronic nicotine administration markedly reduced both the leakage of dye, and brain water content. Chronic treatment with low doses of nicotine Ž0.8 mg kgy1 dayy1 s.c.. lessened the intensity of tonic]clonic seizures observed with a single dose of 3, 5, 8 or 9 mg kgy1 nicotine. The data presented here demonstrate that nicotine pretreatment results in decreased sensitivity to nicotine-induced seizures in rats. Q 1999 Academic Press KEY
WORDS:
nicotine, seizures, blood]brain barrier.
INTRODUCTION Nicotine, a tertiary amine composed of a pyridine and pyrolidine ring, is one of the most widely used substance by humans and has various complex actions. Owing to its lipid solubility it can readily pass into brain parenchyma crossing the blood]brain barrier ŽBBB.. This explains nicotine’s direct effect in the brain, one of which is neuroexcitation. It also operates specific receptors at neuromuscular junctions and in the brain biphasically and has dose-dependent mixed pharmacological effects w1, 2x. It has been shown that this biphasic action is composed of an initial transient stimulation followed by a long lasting depression known as desensitization. Recent in ¨ itro and in ¨ i¨ o investigations have been reported for the neuroprotective effects of nicotine against some excitotoxins w3]5x. High doses of nicotine injection has been also shown to cause hypertension and clonic]tonic convulsions w6, 7x U
Corresponding author.
1043]6618r99r090263]07r$30.00r0
which could both be directly related to microvascular permeability w8, 9x. Previous studies have suggested that nicotineinduced seizure sensitivity decreases as a consequence of nicotine pretreatment w10x. This study was designed to explore the possible beneficial effects of nicotine on vascular functions in the brain and motor convulsions associated with systemic nicotine injection. Vascular effects of nicotine were evaluated by measuring the changes of brain microvascular permeability, and specific gravity ŽSG. which indicates brain water and protein content. MATERIALS AND METHODS Adult male wistar rats ŽDETAM-TURKEY. weighing 200]250 g were used. Animals Ž n s 60. were randomly divided into two groups. One group was acutely treated with seizure-producing doses of nicotines w3, 5, 8 and 9 mg kgy1 i.p, N-3376 Žy. Nicotine free base SIGMAx. Another group of aniQ 1999 Academic Press
Pharmacological Research, Vol. 40, No. 3, 1999
264
mals was pretreated with a low dose of nicotine w11x Ž0.8 mg kgy1 dayy1 i.p.. for 21 days followed by the procedure mentioned in the first group. A control group of rats received either a single injection of the same amount of isotonic saline Ž0.9% NaCl., or daily injection for 21 days. Details of animal preparation and the measurements have been presented previously w2, 6x. Briefly, under light ether anaesthesia, catheters filled with heparinised saline were inserted into the femoral artery and vein and hind quarters of animals were immobilized in a loose-fitting plaster cast. Next the rats were then allowed to recover. The body temperature was monitored with a rectal probe, and external heating lamps were utilized to maintain body temperature at 35]378C. At least 30 min after anaesthesia, when the animals were entirely conscious, 3 ml kgy1 2% Evans blue ŽEB. solution was injected i.v. for visual examination of BBB distribution and spectrophotometric measurements. Ten minutes later seizure-producing doses of nicotine Ž3, 5, 8 and 9 mg kgy1 . were given. The behavioral characteristics of convulsions and duration of convulsions were observed in each individual animal for a period of 30 min. Mean arterial blood pressure was recorded continuously by connecting the arterial catheter to a polygraph system ŽNihon-Kohden, Tokyo, Japan.. At the end of the experiments, 60 min after the injection rats were killed and brains perfused transaortically with a saline solution and removed. After macroscopic evaluation of the brain surface, coronal sections were made and examined for the
extent and the intensity of staining due to EB extravasation. Barrier opening to intravascular EB albumin was graded from 0 Žno staining. to 3 q Ždeep blue. staining. Distribution of EB in different brain regions was also assessed quantitatively using the spectrophotometric method described by Rossner and Temple w12x. In brief, after perfused brains were divided into coronal sections, the wet tissue samples of the six different brain regions were weighed on preweighed aluminium foil, homogenized with 50% trichloroacetic acid in fivefold, centrifuged at 15,000 rpm for 20 min and absorbance was measured at 615 nm. Bilateral specimens of the same eight brain areas from the brain slices were dropped into a gradient column consisting of a heavy and light mixture of kerosene and monobromobenzene. The position which the tissue sample had reached in the column after 5 min was recorded and compared with that of standard K 2 SO4 solutions w13x. Electroencephalographic ŽEEG. recordings were performed in some rats to identify whether a clonic seizure took place and in each animal for the latency of the seizure. The rats in this series were tracheotomized and an artery and a vein were cannulated. The animals were then paralysed with Pavulon ŽOrganonrIstanbul, Turkey. and mechanically ventilated in an 70% N2 and 30% O 2 gas mixture. Xylocaine ŽEczacıbas¸ırIstanbulrTurkey . was employed to infiltrate all surgical wounds. Frontoparietal screw type electrodes were placed through the skull on each side in contact with the meninges.
Table I Mean arterial blood pressure (MABP), blood brain barrier leakage in convulsions induced by various doses of acute nicotine and the effect of chronic nicotine pretreatment Groups (n )
Mean arterial blood pressure (mmHg) Maximum Increase (D P)
Initial Control Ž5. Acute nicotine 3 mg kgy1 Ž5. 5 mg kgy1 Ž5. 8 mg kgy1 Ž5. 9 mg kgy1 Ž5. Choronic nicotine pretreatment q acute nicotine 3 mg kgy1 Ž5. 5 mg kgy1 Ž5. 8 mg kgy1 Ž5. 9 mg kgy1 Ž5. U
100 " 5
]
]
94 " 4 98 " 6 100 " 5 96 "
120 " 5 160 " 8U 165 " 9U 170 " 10U
26 62 65 74
118 " 5 125 " 6UU 123 " 6UU 123 " 6UU
130 " 6 165 " 7 169 " 7 172 " 6
12 UUU 40 UUU 46 UU 48
UUU
Duration (min) Dt
0
q0.5
q1
q2
q3
]
5
]
]
]
]
] ] ] ]
2 ] ] ]
3 3 1 1
] 2 4 3
] ] ] 1
5 4 4 3
] 1 1 2
] ] ] ]
] ] ] ]
] ] ] ]
7 "3 9 "4 10 " 3 12 " 5
2 "0.5UUU 3 "0.5UUU 3 "0.6UUU 4 "1UUU
Degrees of BBB breakdown
Comparison between control values, significance taken as P - 0.01; UU comparison between control onset values, significance taken as P- 0.05; UUU comparison of D P and D t values with acute nicotine groups, significance taken as P - 0.01. 0, no staining; q0.5, local very light blue staining; q1, local light blue staining; q2, generalized moderate staining; q3, generalized deep blue staining.
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Recordings were made on a polygraph system with an amplification sensitivity of 140 mV cmy1 and at a speed of 25 mm sy1 . Statistical analysis was performed using Student’s t-test.
RESULTS Table I lists the mean arterial blood pressure ŽMABP., maximal MABP and the D P in salinetreated controls and rats treated with nicotine. The mean systemic blood pressure in anaesthetized rats at onset was 100 " 5 mmHg in all rats apart from rats treated with low doses of nicotine. Blood pressure increased by approximately 60]70 mmHg and lasted for 7]10 min. The MABP in rats pretreated with low doses of nicotine was held firmly near 125 mmHg. Acute nicotine administration to this group increased MABP by 40]45 mmHg for 2]3 min. Therefore D P and D t were significantly lower than groups treated only with acute nicotine. With acute nicotine injection, spike wave activity and high-voltage potentials were seen in EEG recordings. This activity was maximum in the third
minute and gradually decreased by 10]12 min. The sequence of behavioural changes started with head twitching and clonic movements of forelimbs progressing to generalized tonic]clonic convulsions in 1]2 min Ži.e. latencies . after 3, 5, 8 and 9 mg kgy1 acute injection of nicotine. Low doses of nicotine-pretreated rats were less sensitive to the seizure-producing effect of nicotine than are saline-pretreated rats. Furthermore, rats that are pretreated with nicotine have relatively longer latencies to nicotine-induced seizures with respect to saline-treated controls. Latencies to seizures decreased with increased challenging doses of nicotine. Chronic treatment with low doses of nicotine prevented the occurrence of tonic clonic seizures observed with a single dose of 3, 5, 8 and 9 mg kgy1 nicotine and EEG recordings showed marked attenuation in the frequency and the intensity of spikewaves. In acute nicotine-injected rats, the BBB breakdown to EB was seen and was characteristically always confined to anatomically-limited areas. The regional distribution of BBB breakdown ŽFig. 1. and its intensity are summarized in Table I. With increased challenging doses of nicotine more intense
Fig. 1. legend over page.
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Fig. 1. Spectral analysis of Evans Blue dye in convulsions induced by various doses of acute nicotine and the effects of chronic nicotine pretreatment in cortex, hippocampus, midbrain, striatum, thalamus, cerebellum. mg kgy1A stands for acute nicotine groups, mg kgy1 stands for choronic pretreatment groups receiving acute nicotine. Comparison of chronic pretreatment groups receiving acute nicotine with acute nicotine groups U Significance taken as P - 0.05.
1.0420" 0.55 1.0415" 0.42 1.0424" 0.21 1.0425" 0.35 1.0422" 0.48 1.0450" 0.35 1.0420" 0.43
3 (n s 8)
1.0408" 0.31U 1.0401" 0.35U 1.0415" 0.34 1.0417" 0.34U 1.0423" 0.41 1.0442" 0.39 1.0410" 0.38U
5 (n s 8)
1.0390" 0.19 1.0385" 0.21U 1.0408" 0.19 1.0406" 0.27U 1.0419" 0.29 1.0430" 0.31 1.0395" 0.26U
8 (n s 8)
Acute nicotine (mg kg y 1)
1.0390" 0.27U 1.0383" 0.25U 1.0400" 0.21 1.0405" 0.27U 1.0420" 0.37 1.0428" 0.39 1.0386" 0.41U
9 (n s 8)
1.0448" 0.43 1.0438" 0.33 1.0419" 0.31U 1.0386" 0.25U 1.0381" 0.44
1.0435" 0.49 1.0425" 0.42 1.0430" 0.51 1.0443" 0.45 1.0438" 0.41 1.0465" 0.38 1.0436" 0.38
Control (n s 8)
U
1.0446" 0.39
1.0425" 0.31 1.0425" 0.33 1.0429" 0.37 1.0444" 0.31 1.0438" 0.30 1.0464" 0.34 1.0429" 0.35
21 day chronic nicotine treatment (0.8 mg kg y 1 day y 1) (n s 8) 1.0425" 0.37UU 1.0413" 0.32 1.0428" 0.33 1.0440" 0.34UU 1.0430" 0.36 1.0445" 0.36 1.0430" 0.36UU
5 (n s 8)
1.0421" 0.29UU 1.0415" 0.36 1.0425" 0.33 1.0438" 0.34UU 1.0428" 0.37 1.0447" 0.36 1.0428" 0.34UU
8 (n s 8)
1.0413" 0.31UU 1.0413" 0.31 1.0420" 0.29 1.0437" 0.36UU 1.0428" 0.33 1.0445" 0.35 1.0425" 0.27
9 (n s 8)
1.0445" 0.33UU 1.0440" 0.40UU 1.0437" 0.38UU 1.0435" 0.36UU
1.0430" 0.31 1.0420" 0.34 1.0428" 0.36 1.0445" 0.36UU 1.0436" 0.37 1.0460" 0.38 1.0434" 0.32UU
3 (n s 8)
Acute nicotein (mg kg y 1)
Comparison of chronic pretreatment groups receiving acute nicotine with controls nicotine groups. Significance taken as P - 0.05. UU Comparison of chronic pretreatment groups receiving acute nicotine with acute nicotine groups Significance taken as P - 0.05.
Brain regions Hippocampus Midbrain Hypothalamus Thalamus Striatum Cerebellum Corpus collosum Cortex
Groups
Table II Specific Gravity measurements in convulsions induced by various doses of acute nicotine and the effects of chronic nicotine pretreatment
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BBB breakdown to EB was seen. A 3 q staining was present with 9 mg kgy1 acute nicotine. There was marked difference between BBB interrupting acute nicotine groups with corresponding chronic nicotine pretreatment groups Ž P- 0.051. although, chronic nicotine pretreatment in this study could not completely suppress EB extravasation ŽFig. 1.. SG measurements showed significant differences between control and acute nicotine groups. Corresponding dose groups of cerebellum, cortex, hippocampus Ži.e. Acute ¨ s pretreatment groups. showed significant variations Ž P- 0.05.. There was no significant SG value difference between controls and pretreated groups in midbrain, hypothalamus, caudate nucleus and cerebellum ŽTable II.. Low doses of chronic nicotine administration markedly reduced the leakage of EB and rendered increased SG values reaching to controls ŽFig. 1 and Table II.. Blood]brain barrier opening to EB was consistently present in those experimental groups with a MABP increase at seizure onset ŽTable I..
DISCUSSION
Considerable studies have shown that epileptic seizures may induce neuronal damage and that seizures are accompanied by an abrupt increase in blood pressure and this interacts directly with cerebral blood flow and microvascular permeability w14]17x. In our study, nicotine-induced generalized convulsions were accompanied by elevated MABP scores, in accordance with previous studies w7, 16, 18, 19x. As in many cases, a decreased specific gravity value and regional BBB breakdown Žespecially cortex, hippocampus. were also found w16x. An acute rise in MABP was somewhat smaller, and extravasation of EB was less extensive and SG values were increased in rats treated with a subseizure-producing dose of nicotine. In these rats initial MABP was higher than those of controls before challenging with high doses of nicotine. Since the degree of leakage in epileptic seizures are directly related to the magnitude of systemic blood pressure, it seems that nicotine treatment protected the BBB by limiting the severe elevation of D P. In addition, it is likely that nicotine may alter cerebral blood flow by changing the cerebral circulatory response w6x. Moreover, the administration of nicotine can modify the release of a number of both neurotransmitters and neurohormones w20x. Among the hormones, whose levels are modulated by nicotine, ACTH has been reported that it has to have a role in stimulating the release of steroids such as cortisol from the adrenal cortex w21x.
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A relatively new aspect of nicotine’s action has been put forward by Owman et al. w22x. In their studies they have employed nicotine pretreatment on rats whose meso-striatal dopamine system had been transected. They have found amelioration in blood flow and glucose utilization in caudate putamen on the side where meso-striatal dopamine system had been transected. These observations and our previous studies which demonstrated that the administration of glucocorticoids produced a decreased cerebrovascular permeability, while withdrawal of the drug resulted in increased permeability w23, 24x which makes it possible to speculate that the pituitary]adrenal axis might also play an important role in the regulation of permeability of brain microvasculature in nicotine-pretreated animals. It is well accepted that low doses of nicotine interacts biphasically with specific receptors in the brain. This biphasic action is similar to an initial transient stimulation followed by a more long lasting depression known as desensitization w10x. At higher doses nicotine has been shown to cause clonic]tonic convulsions w1, 7, 19x. De Fiebre et al. w10x showed that nicotine pretreatment rendered animals less susceptible to nicotine-induced seizures than salinepretreated controls. The data presented here clearly demonstrated that nicotine-pretreated animals are less sensitive to the seizure producing effects of nicotine than saline-pretreated rats. Furthermore, rats that are pretreated with nicotine have significantly longer latencies to nicotine-induced seizures. One possible explanation for both the decreased sensitivity and longer latencies is nicotinic-receptor desensitisation or inactivation in CNS caused by low-dose nicotine treatment. Indeed, Simoska et al. w25x have shown that desensitisation and inactivation occur in nicotinic receptors after treatment with nicotine agonists. In addition, decreased sensitivity to seizures reported here could also be caused by a number of other factors such as an active metabolite of nicotine Ži.e. nicotine N-oxide. by inhibiting nicotine’s action as suggested Barras et al. w26x. Finally this work showed that nicotine pretreatment may have protective actions on neuronal and vascular functions in the brain against nicotineinduced seizures, shown by normalized EEG activity in addition to increased SG values Ži.e. getting closer to control SG values. and decreased EB extravasation Žby spectrophotometry.. This protective effect could be explained by the nicotine-induced desensitization or inactivation of the nicotinic receptors in the CNS. The observed changes indicating the protective effects in the permeability of BBB and specific gravity, may be partially due to secondary metabolic activation of nicotine on the cerebral microenvironment.
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ACKNOWLEDGEMENTS This work was supported by the Research Fund of the University of Istanbul 660-210994.
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