- Email: [email protected]
Effect of acetyl-l -carnitine on hyperactivity and spatial memory deficits of rats exposed to neonatal anoxia
Neuroscience Letters 223 (1997) 201–205
Effect of acetyl-l-carnitine on hyperactivity and spatial memory deficits of rats exposed to neonatal anoxia Elisabetta Dell’Anna a ,*, Laura Iuvone b, Stefano Calzolari c, Maria Concetta Geloso d a
Department of Experimental and Clinical Pathology and Medicine, Chair of Neurology, University of Udine, Piazza S. Maria della Misericordia, 1, I-33100 Udine, Italy b Institute of Neurology, Catholic University, Rome, Italy c Child Neuropsychiatry Unit, Trento, Italy d Institute of Histology and Embryology, Catholic University, Rome, Italy Received 18 November 1996; revised version received 21 January 1997; accepted 23 January 1997
Abstract The effect of acetyl-l-carnitine (ALC) on behavioral deficits following neonatal anoxia (N2 100% for 25 min at 30 h after birth) was studied in the rat. Transient hyperactivity at P20–P45 postnatal days and permanent spatial memory deficits were shown by anoxic rats. A chronic ALC treatment (50 mg/kg per die injected intraperitoneally from P2, after anoxia, to P60) significantly reduced the transient increase in sniffing, rearing and locomotory activity of anoxic rats, but, mostly, ameliorated the spatial memory performances in a maze at P30–P40 and in a water maze at P50–P60. No behavioral changes were seen in ALC-treated animals that received sham-exposure at birth. On the basis of these results, the use of ALC for the treatment of perinatal asphyctic insults in children is suggested. 1997 Elsevier Science Ireland Ltd. Keywords: Perinatal asphyxia; Attention deficit-hyperactivity disorder; Acetyl-l-carnitine; Hyperactivity; Spatial memory; Neuroprotection
Impairment of oxygen/energy supply during the perinatal period may affect neuronal functions and induce cell death. Brain damage after perinatal anoxia or asphyxia depends upon several factors, but duration and severity of the insult, as well as maturation and metabolic stage at which the various brain regions are found, appear the most critical [17]. Thus, when death of the newborn is not occurring following perinatal anoxia or asphyxia, various neurological deficits including hyperactivity, learning disabilities, mental retardation, epilepsy, cerebral palsy, or dystonia, may develop both in humans [1,14,21] and in experimental animals [7,13]. Treatment of these disorders should consider not only therapy for the different symptoms but, primarily, intervention on mechanisms involved in their pathogenesis. Neuronal cell changes after anoxic or ischemic lesions result from a complex series of events that are triggered * Corresponding author. Tel.: +39 432 559827; fax: +39 432 42097; e-mail: [email protected]
during the oxygen deprivation phase, and persist during the subsequent reoxygenation/recirculation period. Disruption of energy metabolism, activation of excitatory aminoacids, alteration in intra-extracellular ionic balance, abnormal intracellular calcium influx, accumulation of lactic acid, excessive production of free radicals, have a primary role in the cascade of cytotoxic processes [3,4,24]. Therefore, substances contrasting the deleterious effects of oxygen/energy impairment might counteract the development of functional alterations following perinatal asphyctic insults. Acetyl-l-carnitine (ALC) is receiving attention as an effective drug in neuroprotection [3,12]. ALC is an acetyl derivate of carnitine, detectable in various brain regions since early phases of development [18], that is required for transport and utilization of fatty acids [22] and in energy metabolism [3,6,18]. Thus, we investigated the effect of ALC treatment on the behavioral deficits induced by neonatal anoxia in a rat model that has been characterized in our laboratory in some of its behavioral [7], neuroanato-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )1 3411-5
202
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
mical [7,9] and neurochemical aspects [8,10]. In the present study, the activity in open field and in maze tests was analyzed in anoxic and sham-treated rats that received ALC immediately after anoxia until adulthood. Eight litters of Wistar rats (Catholic University Animal Department, Rome, Italy), each consisting of 9–10 pups, were used. The pups were randomly assigned to anoxia or sham-treatment. Anoxia was induced at approximately 30 h after birth (postnatal day 2, P2) by exposure to nitrogen 100% (N2) for 25 min, at 2.7 atm., with a flow of 9 l/min; during the experiment, the temperature was kept at +35°C, by immersing the glass chamber, where the pups were placed, in a water bath. Sham-treatment consisted of exposing pups to room air when in the chamber. Approximately 10% mortality was observed immediately after the N2 exposure [7]. Following treatment, groups of five pups were returned to their own mother until weaning, at P21. The animals were housed in air conditioned rooms with controlled temperature (22–24°C) and dark/light cycle (10:14 h). Animals were randomly assigned to ALC or saline treatment. Groups of 10 sham-treated (S) and 10 anoxic (A) rats received an intraperitoneal injection of a 0.5 ml volume of either ALC (Nicetile, Sigma Tau S.p.A., Pomezia, Italy; 50 mg/kg per die dissolved in saline; groups SN and AN) or saline (groups SS and AS). The injection was given daily, at 0900–1000 h, starting at P2 1 h after anoxia or sham exposure, until P60. Spontaneous behavior in open field was evaluated every fifth day, from P5 to P60. Couples of animals, belonging to the same group, were placed for 10 min in an open field, at 1500–1700 h. The sessions were videorecorded and subsequently analyzed for sniffing (number of sniffing movements in the air), rearing (number of rearing up on hind legs) and locomotory activity (time spent moving). At P30, P35 and P40 the rats were tested, twice a day, in a maze with food as a reward (for detailed description, see [7]). Thirty-six hours before every first daily trial and for other 4 h until completion of the second trial, the animals were kept deprived of food. In all trials, the rat was put in the South-West sector and it had to reach the food pellet located in the North-East sector, without any other reinforcement. The latency to find the food was recorded with a stopwatch. From P50 to P60 the animals were tested in a water maze. Every day, a task consisting of four consecutive trials was run, for a total of 11 tasks. In all trials, the rat was placed in the same starting location (West), facing the wall of the pool, and it had to reach, without any reinforcement, a platform which was hidden (Place Navigation; phase I, tasks 1–6, NE quadrant; phase II, tasks 10–11, SE quadrant) or left visible (Cue Navigation, tasks 7–9, SE quadrant). The rat was removed from the water and placed on the platform for 30 s if it failed to locate the platform after the cut-off time of 120 s. The latency to reach the platform was recorded with a stopwatch. The results
obtained by individual animals in the four trials of each task were averaged to constitute one observation. A three-way analysis of variance (ANOVA) was used to test the effect of neonatal and drug treatment, day of observation (for sniffing, rearing and locomotory activity in the open field) or task (for maze and water maze tests), and their interactions. The three phases of navigation of the water maze test were analyzed separately. Post-hoc comparisons were done with Fisher test, when appropriate, at a significance level of P , 0.05, for the two-tailed test. After completion of the behavioral procedures, the animals were transcardially perfused, under deep barbiturate anesthesia, with 10% buffered formalin. Sagittal 20 mm serial sections were cut from the brain and processed for thionin staining. The sections were analyzed under light microscopy for morphological changes and for evaluation of cell density in the hippocampus [7,9]. Physical growth and development were similar in all experimental groups. No side effect of ALC treatment could be seen. Histological analysis of the brain did not show changes that could be related to ALC treatment (data not shown); also, the effect of neonatal anoxia on hippocampal cell density previously reported [7,9], did not appear to be modified by ALC treatment (data not shown). Neonatal and drug treatment as well as the day of observation significantly influenced sniffing (treatment, F = 54.107, P , 0.001; testing day, F = 54.43, P , 0.001; interaction, F = 5.81, P , 0.001), rearing (treatment, F = 67.78, P , 0.001; testing day, F = 54.43, P , 0.001; interaction, F = 5.81, P , 0.001) and locomotory activity (treatment, F = 106.567, P , 0.001; testing day, F = 38.755, P , 0.001; interaction, F = 3.432, P , 0.001). A progressive increase in the number of sniffing movements was present in all groups from P5 to P20, but a further increment until P40 was observed only in anoxic animals that did not receive ALC (group AS). During this period (P20–P40) the number of sniffing movements was significantly higher in the AS group, compared to shamtreated (SS, SN) animals; also, it appeared significantly increased with respect to that of anoxic-ALC treated (AN) animals from P30 to P40. At the P45–P60 observations no differences among experimental groups could be found (Fig. 1). The number of rearing movements increased until P45 in all animals, but the AS values were significantly higher than the SS and SN ones from P25 to P45; at P45 a significant difference between AS and AN animals was also found (Fig. 1). Sham-treated animals (SS and SN) presented a progressive increase in locomotory activity until P25, while anoxic rats (AS and AN) until P40. Significant differences between AS and SS or SN groups were seen at P35, P40 and P45; at P45, an effect of ALC treatment on locomotory activity of anoxic animals was also found. At the P50–P60 observations no differences among groups could be seen (Fig. 1).
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
203
among groups could be seen during cue navigation (Fig. 2). Following neonatal anoxia, rats presented a transient increase in sniffing, rearing and locomotory activity in open field at the P25–P45 observations; during this period, abnormal performances in the maze test were also evident, indicating, as discussed in a previous paper [7], spatial memory deficits. Alteration in spatial abilities were also present in anoxic rats in the water maze test at P50–P60, which was a period when their open field behavior did not differ from that of sham-treated animals. Thus, the results obtained in the present study confirm previous observations in this model [7] that neonatal anoxia induces transitory hyperactivity and permanent spatial memory deficits, as the latter have been detected until P120 (Dell’Anna and Iuvone, unpublished observations). Similarly, cognitive and behavioral disorders may persist for a long time in humans that suffered mild perinatal asphyctic insults [1,7,21]. The behavioral disturbances seen in rats are associated with neuroanatomical and neurochemical changes in the hippocampus, as neuronal cell loss in the CA1 region [7,9], reduction of GABA-immunoreactive neurons [10] and altered monoamine systems maturation [8] have been observed.
Fig. 1. Sniffing (upper graph), rearing (middle graph) and locomotory activity (lower graph) in open field shown by sham-treated (S) and anoxic (A) rats treated with saline (S) or ALC 50 mg/kg per die (N) (see legend) at the P5–P60 postnatal period. Data are presented as means ± SEM. Asterisks above the AS line indicate significant differences of AS vs. SS and SN groups; asterisks above the AN line indicate significant differences between AS and AN groups.
In the maze test, a progressive decrease in the time needed to reach the food during subsequent trials was present in all groups (F = 17.91, P , 0.001); however, AS rats used significantly longer latency than SS, SN and AN animals at each trial (F = 25.746, P , 0.001) (Fig. 2). In the water maze test, sham-treated animals (SS and SN) reduced progressively their latency to find a hidden platform during subsequent trials of the first place navigation phase. A further reduction in the latency was seen during cue navigation and in the second place navigation phase, when they reached directly the platform. In both place navigation phases (phase I, F = 1.856, P , 0.01; phase II, F = 3.964, P , 0.02), AS rats required longer latencies than SS, SN or AN animals, while no differences
Fig. 2. Latency exhibited by sham-treated (S) and anoxic (A) rats treated with saline (S) or ALC (N) (see legend), to reach the food (Maze test) or the platform (Water maze test) in the various tasks of the tests. Values are presented as means ± SEM. Asterisks indicate significant differences of AS vs. SS, SN and AN groups.
204
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
A chronic ALC treatment was established from P2, after anoxia exposure, to P60, in order to test a possible therapeutic role of this drug on the behavioral consequences of neonatal anoxia. Although marked effects on lactic acidosis and brain energy production have been seen after acute ALC treatment following ischemia [3], a chronic schedule was used here based on observations that post-anoxic neuronal damage occurs either immediately or with some delay after the insult [7,9,11] and that mitochondrial function may be persistently damaged even after rapid correction of oxygen supply [3]. The dose of 50 mg/kg per die was chosen since this has been demonstrated to be the threshold for inducing significant striatal or hippocampal release of acetylcholine [15] and to be efficacious, similarly to higher doses, in reducing behavioral alterations during aging [16]. No side effects of ALC treatment could be seen in any animal. No modification in open field or maze behavior was found in sham-treated rats which received ALC compared to those who were injected with saline or that did not receive any treatment [7], as also reported by other studies in normal young animals [5,16], indicating that ALC should not influence normal behavior. In anoxic rats, open field activity was reduced by ALC during the P30–P45 period; in particular, the sniffing behavior was the most affected by the treatment, while a minor effect, restricted to the P45 observation, was present on rearing and locomotion. It is possible that sniffing is a behavioral parameter more sensitive to toxic, pharmachological and environmental influences [7]. A marked improvement of spatial memory deficits was seen under ALC treatment, and in all tasks of both mazes anoxic ALC-treated animals showed significantly shorter latencies to reach the goal than the anoxic untreated rats. However, no changes were seen in the cue navigation phase of the water maze, where anoxic rats displayed a normal behavior since it does not require spatial information recall [7]. It has been reported that ALC reduces changes in locomotory and rearing activity [5,16] as well as deficits in passive and active avoidance of aged rats [2,25]; in humans, cognitive deficits in Alzheimer-type dementia were ameliorated by ALC [20,23]. The effect of ALC treatment on behavioral and cognitive functions has been generally related to its acetylcholine-like activity [2,6], as there is evidence that ALC influences acetylcholine release and activity [15]. Indeed, changes in cholinergic neurotransmission in the hippocampus have been observed in this model of neonatal anoxia [13], and they could be involved in the behavioral disturbances described. However, a neuroprotective and neurotrophic action of ALC has been suggested, that could counteract the development of functional alterations following anoxic neuronal insults. In in vitro experiments ALC was effective in protecting cells from glutamate, kainic acid and beta amyloid fragment 25–35 toxicity [12]; furthermore, increase in respiratory activity [6], restoration of energy production [3], reduction of lactic acid levels [3] and activation of protein
kinase C [19] occurred after ALC treatment following different experimental conditions including ischemia [3]; finally, NGF levels were increased by ALC [2], suggesting a neurotrophic effect. However, at the histological level, the morphological changes induced by neonatal anoxia [7,9] were unmodified by ALC treatment, at least in the brain region analyzed (hippocampus) and with the method utilized. In conclusion, ALC treatment following neonatal anoxia results effective on both hyperactivity and spatial memory deficits; this finding suggests to extend the clinical use of ALC in perinatal asphyctic insults in children. [1] Amiel-Tison, C. and Ellison, P., Birth asphyxia in the fullterm newborn: early assessment and outcome, Dev. Med. Child Neurol., 28 (1986) 671–682. [2] Angelucci, L., Ramacci, M.T., Taglialatela, G., Hulsebosh, C., Morgan, B., Werrbach-Perez, K. and Perez-Polo, R., Nerve growth factor binding in aged rat central nervous system: effect of acetyll-carnitine, J. Neurosci. Res., 20 (1988) 491–496. [3] Aureli, T., Miccheli, T., Di Cocco, E.M., Ghirardi, O., Giuliani, A., Ramacci, M.T. and Conti, F., Effect of acetyl-l-carnitine on recovery of brain phosphorus metabolites and lactic acid level during reperfusion after cerebral ischemia in the rat – study by 13P and 1HNMR spectroscopy, Brain Res., 643 (1994) 92–99. [4] Barks, J.D.E. and Silverstein, F.S., Excitatory amino acids contribute to the pathogenesis of perinatal hypoxic-ischemic injury, Brain Pathol., 2 (1992) 235–243. [5] Blokland, A., Raaijmakers, W., van der Staay, J.F. and Jolles, J., Differential effect of acetyl-l-carnitine on open field behavior in young and old rats, Physiol. Behav., 47 (1990) 783–785. [6] Curti, D., Dagani, F., Galmozzi, M.R. and Marzatico, F., Effect of aging and acetyl-l-carnitine on energetic and cholinergic metabolism in rat brain regions, Mech. Aging Dev., 47 (1989) 39–45. [7] Dell’Anna, M.E., Calzolari, S., Molinari, M., Iuvone, L. and Calimici, R., Neonatal anoxia induces transitory hyperactivity, permanent spatial memory deficits and CA1 cell density reduction in developing rats, Behav. Brain Res., 45 (1991) 125–134. [8] Dell’Anna, M.E., Luthman, J., Lindqvist, E. and Olson, L., Development of monoamine systems after neonatal anoxia in rats, Brain Res. Bull., 32 (1993) 159–170. [9] Dell’Anna, M.E., Geloso, M.C., Draisci, G. and Luthman, J., Transient changes in fos and GFAp immunoreactivity precede neuronal cell loss in the rat hippocampus following neonatal anoxia, Exp. Neurol., 131 (1995) 144–156. [10] Dell’Anna, E., Geloso, M.C., Magarelli, M. and Molinari, M., Development of gamma-aminobutyric acid (GABA) and calcium binding proteins immunoreactivity in the rat hippocampus following neonatal anoxia, Neurosci. Lett., 211 (1996) 93–96. [11] Filloux, F.M., Adair, J. and Narang, N., The temporal evolution of striatal dopamine receptor binding and mRNA expression following hypoxia-ischemia in the neonatal rat, Dev. Brain Res., 94 (1996) 81–91. [12] Forloni, G., Angeretti, N. and Smiroldo, S., Neuroprotective activity of acetyl-l-carnitine: studies in vitro, J. Neurosci. Res., 37 (1994) 92–96. [13] Hershkowitz, M., Grimm, V. and Speiser, Z., The effects of postnatal anoxia on behavior and on muscarinic and beta-adrenergic receptors in the hippocampus of the developing rats, Dev. Brain Res., 7 (1983) 147–155. [14] Hill, A. and Volpe, J.J., Seizures, hypoxic-ischemic injury and intraventricular hemorrhage in the newborn, Ann. Neurol., 10 (1981) 109–121. [15] Imperato, A., Ramacci, M.T. and Angelucci, L., Acetyl-l-carnitine
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
[16]
[17]
[18]
[19]
[20]
enhances acetylcholine release in the striatum and hippocampus of awake freely moving rats, Neurosci. Lett., 107 (1989) 251–255. Kohjimoto, Y., Ogawa, T., Matsumoto, M., Shirakawa, K., Kuwaki, T., Yasuda, H., Anami, K., Fuji, T., Satoh, H. and Ono, T., Effects of acetyl-l-carnitine on the brain lipofuscin content and emotional behavior in aged rats, Jpn. J. Pharmacol., 48 (1988) 365– 371. Lipton, S.A. and Kater, S.B., Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, Trends Neurosci., 12 (1989) 265–270. Morris, A.J. and Carey, E.M., Postnatal changes in the concentration of carnitine and acetylcarnitine in the rat brain, Dev. Brain Res., 8 (1983) 381–384. Pascale, A., Milano, S., Corsico, N., Lucchi, L., Battaini, F., Arrigoni Martelli, E., Trabucchi, M. and Govoni, S., Protein kinase C activation and antiamnesic effect of acetyl-l-carnitine: in vitro and in vivo studies, Eur. J. Pharmacol., 265 (1994) 1–7. Pettegrew, J.W., Klunk, W.E., Kanagasabai, P., Kanfler, J.N. and McClure, R.J., Clinical and neurochemical effects of acetyl-l-carnitine in Alzheimer’s disease, Neurobiol. Aging, 16 (1995) 1–4.
205
[21] Shaffer, S.Q., Shaffer, D., O’Connor, P.A. and Stockman, C.J., Hard thought on neurological ‘soft signs’. In M. Rutter (Ed.), Developmental Neuropsychiatry, Guilford Press, New York, 1983, pp. 133–143. [22] Siliprandi, N., Siliprandi, D. and Ciman, M., Stimulation of oxidation of mitochondrial fatty acids and of acetate by acetylcarnitine, Biochem. J., 96 (1965) 777–782. [23] Spagnoli, A., Lucca, U., Menasce, G., Bandera, L., Cizza, G., Forloni, G. and Tettamanti, M. et al., Long-term acetyl-l-carnitine treatment in Alzheimer’s disease, Neurology, 41 (1991) 1726– 1732. [24] Szatkowski, M. and Attwell, D., Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms, Trends Neurosci., 17 (1994) 359–365. [25] Valerio, C., Clementi, G., Spadaro, F., D’Agata, V., Raffaele, R., Grassi, M., Lauria, N. and Drago, F., The effects of acetyl-l-carnitine on experimental models of learning and memory deficits in the old rat, Funct. Neurol., 4 (1989) 387–390.
Effect of acetyl-l-carnitine on hyperactivity and spatial memory deficits of rats exposed to neonatal anoxia Elisabetta Dell’Anna a ,*, Laura Iuvone b, Stefano Calzolari c, Maria Concetta Geloso d a
Department of Experimental and Clinical Pathology and Medicine, Chair of Neurology, University of Udine, Piazza S. Maria della Misericordia, 1, I-33100 Udine, Italy b Institute of Neurology, Catholic University, Rome, Italy c Child Neuropsychiatry Unit, Trento, Italy d Institute of Histology and Embryology, Catholic University, Rome, Italy Received 18 November 1996; revised version received 21 January 1997; accepted 23 January 1997
Abstract The effect of acetyl-l-carnitine (ALC) on behavioral deficits following neonatal anoxia (N2 100% for 25 min at 30 h after birth) was studied in the rat. Transient hyperactivity at P20–P45 postnatal days and permanent spatial memory deficits were shown by anoxic rats. A chronic ALC treatment (50 mg/kg per die injected intraperitoneally from P2, after anoxia, to P60) significantly reduced the transient increase in sniffing, rearing and locomotory activity of anoxic rats, but, mostly, ameliorated the spatial memory performances in a maze at P30–P40 and in a water maze at P50–P60. No behavioral changes were seen in ALC-treated animals that received sham-exposure at birth. On the basis of these results, the use of ALC for the treatment of perinatal asphyctic insults in children is suggested. 1997 Elsevier Science Ireland Ltd. Keywords: Perinatal asphyxia; Attention deficit-hyperactivity disorder; Acetyl-l-carnitine; Hyperactivity; Spatial memory; Neuroprotection
Impairment of oxygen/energy supply during the perinatal period may affect neuronal functions and induce cell death. Brain damage after perinatal anoxia or asphyxia depends upon several factors, but duration and severity of the insult, as well as maturation and metabolic stage at which the various brain regions are found, appear the most critical [17]. Thus, when death of the newborn is not occurring following perinatal anoxia or asphyxia, various neurological deficits including hyperactivity, learning disabilities, mental retardation, epilepsy, cerebral palsy, or dystonia, may develop both in humans [1,14,21] and in experimental animals [7,13]. Treatment of these disorders should consider not only therapy for the different symptoms but, primarily, intervention on mechanisms involved in their pathogenesis. Neuronal cell changes after anoxic or ischemic lesions result from a complex series of events that are triggered * Corresponding author. Tel.: +39 432 559827; fax: +39 432 42097; e-mail: [email protected]
during the oxygen deprivation phase, and persist during the subsequent reoxygenation/recirculation period. Disruption of energy metabolism, activation of excitatory aminoacids, alteration in intra-extracellular ionic balance, abnormal intracellular calcium influx, accumulation of lactic acid, excessive production of free radicals, have a primary role in the cascade of cytotoxic processes [3,4,24]. Therefore, substances contrasting the deleterious effects of oxygen/energy impairment might counteract the development of functional alterations following perinatal asphyctic insults. Acetyl-l-carnitine (ALC) is receiving attention as an effective drug in neuroprotection [3,12]. ALC is an acetyl derivate of carnitine, detectable in various brain regions since early phases of development [18], that is required for transport and utilization of fatty acids [22] and in energy metabolism [3,6,18]. Thus, we investigated the effect of ALC treatment on the behavioral deficits induced by neonatal anoxia in a rat model that has been characterized in our laboratory in some of its behavioral [7], neuroanato-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )1 3411-5
202
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
mical [7,9] and neurochemical aspects [8,10]. In the present study, the activity in open field and in maze tests was analyzed in anoxic and sham-treated rats that received ALC immediately after anoxia until adulthood. Eight litters of Wistar rats (Catholic University Animal Department, Rome, Italy), each consisting of 9–10 pups, were used. The pups were randomly assigned to anoxia or sham-treatment. Anoxia was induced at approximately 30 h after birth (postnatal day 2, P2) by exposure to nitrogen 100% (N2) for 25 min, at 2.7 atm., with a flow of 9 l/min; during the experiment, the temperature was kept at +35°C, by immersing the glass chamber, where the pups were placed, in a water bath. Sham-treatment consisted of exposing pups to room air when in the chamber. Approximately 10% mortality was observed immediately after the N2 exposure [7]. Following treatment, groups of five pups were returned to their own mother until weaning, at P21. The animals were housed in air conditioned rooms with controlled temperature (22–24°C) and dark/light cycle (10:14 h). Animals were randomly assigned to ALC or saline treatment. Groups of 10 sham-treated (S) and 10 anoxic (A) rats received an intraperitoneal injection of a 0.5 ml volume of either ALC (Nicetile, Sigma Tau S.p.A., Pomezia, Italy; 50 mg/kg per die dissolved in saline; groups SN and AN) or saline (groups SS and AS). The injection was given daily, at 0900–1000 h, starting at P2 1 h after anoxia or sham exposure, until P60. Spontaneous behavior in open field was evaluated every fifth day, from P5 to P60. Couples of animals, belonging to the same group, were placed for 10 min in an open field, at 1500–1700 h. The sessions were videorecorded and subsequently analyzed for sniffing (number of sniffing movements in the air), rearing (number of rearing up on hind legs) and locomotory activity (time spent moving). At P30, P35 and P40 the rats were tested, twice a day, in a maze with food as a reward (for detailed description, see [7]). Thirty-six hours before every first daily trial and for other 4 h until completion of the second trial, the animals were kept deprived of food. In all trials, the rat was put in the South-West sector and it had to reach the food pellet located in the North-East sector, without any other reinforcement. The latency to find the food was recorded with a stopwatch. From P50 to P60 the animals were tested in a water maze. Every day, a task consisting of four consecutive trials was run, for a total of 11 tasks. In all trials, the rat was placed in the same starting location (West), facing the wall of the pool, and it had to reach, without any reinforcement, a platform which was hidden (Place Navigation; phase I, tasks 1–6, NE quadrant; phase II, tasks 10–11, SE quadrant) or left visible (Cue Navigation, tasks 7–9, SE quadrant). The rat was removed from the water and placed on the platform for 30 s if it failed to locate the platform after the cut-off time of 120 s. The latency to reach the platform was recorded with a stopwatch. The results
obtained by individual animals in the four trials of each task were averaged to constitute one observation. A three-way analysis of variance (ANOVA) was used to test the effect of neonatal and drug treatment, day of observation (for sniffing, rearing and locomotory activity in the open field) or task (for maze and water maze tests), and their interactions. The three phases of navigation of the water maze test were analyzed separately. Post-hoc comparisons were done with Fisher test, when appropriate, at a significance level of P , 0.05, for the two-tailed test. After completion of the behavioral procedures, the animals were transcardially perfused, under deep barbiturate anesthesia, with 10% buffered formalin. Sagittal 20 mm serial sections were cut from the brain and processed for thionin staining. The sections were analyzed under light microscopy for morphological changes and for evaluation of cell density in the hippocampus [7,9]. Physical growth and development were similar in all experimental groups. No side effect of ALC treatment could be seen. Histological analysis of the brain did not show changes that could be related to ALC treatment (data not shown); also, the effect of neonatal anoxia on hippocampal cell density previously reported [7,9], did not appear to be modified by ALC treatment (data not shown). Neonatal and drug treatment as well as the day of observation significantly influenced sniffing (treatment, F = 54.107, P , 0.001; testing day, F = 54.43, P , 0.001; interaction, F = 5.81, P , 0.001), rearing (treatment, F = 67.78, P , 0.001; testing day, F = 54.43, P , 0.001; interaction, F = 5.81, P , 0.001) and locomotory activity (treatment, F = 106.567, P , 0.001; testing day, F = 38.755, P , 0.001; interaction, F = 3.432, P , 0.001). A progressive increase in the number of sniffing movements was present in all groups from P5 to P20, but a further increment until P40 was observed only in anoxic animals that did not receive ALC (group AS). During this period (P20–P40) the number of sniffing movements was significantly higher in the AS group, compared to shamtreated (SS, SN) animals; also, it appeared significantly increased with respect to that of anoxic-ALC treated (AN) animals from P30 to P40. At the P45–P60 observations no differences among experimental groups could be found (Fig. 1). The number of rearing movements increased until P45 in all animals, but the AS values were significantly higher than the SS and SN ones from P25 to P45; at P45 a significant difference between AS and AN animals was also found (Fig. 1). Sham-treated animals (SS and SN) presented a progressive increase in locomotory activity until P25, while anoxic rats (AS and AN) until P40. Significant differences between AS and SS or SN groups were seen at P35, P40 and P45; at P45, an effect of ALC treatment on locomotory activity of anoxic animals was also found. At the P50–P60 observations no differences among groups could be seen (Fig. 1).
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
203
among groups could be seen during cue navigation (Fig. 2). Following neonatal anoxia, rats presented a transient increase in sniffing, rearing and locomotory activity in open field at the P25–P45 observations; during this period, abnormal performances in the maze test were also evident, indicating, as discussed in a previous paper [7], spatial memory deficits. Alteration in spatial abilities were also present in anoxic rats in the water maze test at P50–P60, which was a period when their open field behavior did not differ from that of sham-treated animals. Thus, the results obtained in the present study confirm previous observations in this model [7] that neonatal anoxia induces transitory hyperactivity and permanent spatial memory deficits, as the latter have been detected until P120 (Dell’Anna and Iuvone, unpublished observations). Similarly, cognitive and behavioral disorders may persist for a long time in humans that suffered mild perinatal asphyctic insults [1,7,21]. The behavioral disturbances seen in rats are associated with neuroanatomical and neurochemical changes in the hippocampus, as neuronal cell loss in the CA1 region [7,9], reduction of GABA-immunoreactive neurons [10] and altered monoamine systems maturation [8] have been observed.
Fig. 1. Sniffing (upper graph), rearing (middle graph) and locomotory activity (lower graph) in open field shown by sham-treated (S) and anoxic (A) rats treated with saline (S) or ALC 50 mg/kg per die (N) (see legend) at the P5–P60 postnatal period. Data are presented as means ± SEM. Asterisks above the AS line indicate significant differences of AS vs. SS and SN groups; asterisks above the AN line indicate significant differences between AS and AN groups.
In the maze test, a progressive decrease in the time needed to reach the food during subsequent trials was present in all groups (F = 17.91, P , 0.001); however, AS rats used significantly longer latency than SS, SN and AN animals at each trial (F = 25.746, P , 0.001) (Fig. 2). In the water maze test, sham-treated animals (SS and SN) reduced progressively their latency to find a hidden platform during subsequent trials of the first place navigation phase. A further reduction in the latency was seen during cue navigation and in the second place navigation phase, when they reached directly the platform. In both place navigation phases (phase I, F = 1.856, P , 0.01; phase II, F = 3.964, P , 0.02), AS rats required longer latencies than SS, SN or AN animals, while no differences
Fig. 2. Latency exhibited by sham-treated (S) and anoxic (A) rats treated with saline (S) or ALC (N) (see legend), to reach the food (Maze test) or the platform (Water maze test) in the various tasks of the tests. Values are presented as means ± SEM. Asterisks indicate significant differences of AS vs. SS, SN and AN groups.
204
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
A chronic ALC treatment was established from P2, after anoxia exposure, to P60, in order to test a possible therapeutic role of this drug on the behavioral consequences of neonatal anoxia. Although marked effects on lactic acidosis and brain energy production have been seen after acute ALC treatment following ischemia [3], a chronic schedule was used here based on observations that post-anoxic neuronal damage occurs either immediately or with some delay after the insult [7,9,11] and that mitochondrial function may be persistently damaged even after rapid correction of oxygen supply [3]. The dose of 50 mg/kg per die was chosen since this has been demonstrated to be the threshold for inducing significant striatal or hippocampal release of acetylcholine [15] and to be efficacious, similarly to higher doses, in reducing behavioral alterations during aging [16]. No side effects of ALC treatment could be seen in any animal. No modification in open field or maze behavior was found in sham-treated rats which received ALC compared to those who were injected with saline or that did not receive any treatment [7], as also reported by other studies in normal young animals [5,16], indicating that ALC should not influence normal behavior. In anoxic rats, open field activity was reduced by ALC during the P30–P45 period; in particular, the sniffing behavior was the most affected by the treatment, while a minor effect, restricted to the P45 observation, was present on rearing and locomotion. It is possible that sniffing is a behavioral parameter more sensitive to toxic, pharmachological and environmental influences [7]. A marked improvement of spatial memory deficits was seen under ALC treatment, and in all tasks of both mazes anoxic ALC-treated animals showed significantly shorter latencies to reach the goal than the anoxic untreated rats. However, no changes were seen in the cue navigation phase of the water maze, where anoxic rats displayed a normal behavior since it does not require spatial information recall [7]. It has been reported that ALC reduces changes in locomotory and rearing activity [5,16] as well as deficits in passive and active avoidance of aged rats [2,25]; in humans, cognitive deficits in Alzheimer-type dementia were ameliorated by ALC [20,23]. The effect of ALC treatment on behavioral and cognitive functions has been generally related to its acetylcholine-like activity [2,6], as there is evidence that ALC influences acetylcholine release and activity [15]. Indeed, changes in cholinergic neurotransmission in the hippocampus have been observed in this model of neonatal anoxia [13], and they could be involved in the behavioral disturbances described. However, a neuroprotective and neurotrophic action of ALC has been suggested, that could counteract the development of functional alterations following anoxic neuronal insults. In in vitro experiments ALC was effective in protecting cells from glutamate, kainic acid and beta amyloid fragment 25–35 toxicity [12]; furthermore, increase in respiratory activity [6], restoration of energy production [3], reduction of lactic acid levels [3] and activation of protein
kinase C [19] occurred after ALC treatment following different experimental conditions including ischemia [3]; finally, NGF levels were increased by ALC [2], suggesting a neurotrophic effect. However, at the histological level, the morphological changes induced by neonatal anoxia [7,9] were unmodified by ALC treatment, at least in the brain region analyzed (hippocampus) and with the method utilized. In conclusion, ALC treatment following neonatal anoxia results effective on both hyperactivity and spatial memory deficits; this finding suggests to extend the clinical use of ALC in perinatal asphyctic insults in children. [1] Amiel-Tison, C. and Ellison, P., Birth asphyxia in the fullterm newborn: early assessment and outcome, Dev. Med. Child Neurol., 28 (1986) 671–682. [2] Angelucci, L., Ramacci, M.T., Taglialatela, G., Hulsebosh, C., Morgan, B., Werrbach-Perez, K. and Perez-Polo, R., Nerve growth factor binding in aged rat central nervous system: effect of acetyll-carnitine, J. Neurosci. Res., 20 (1988) 491–496. [3] Aureli, T., Miccheli, T., Di Cocco, E.M., Ghirardi, O., Giuliani, A., Ramacci, M.T. and Conti, F., Effect of acetyl-l-carnitine on recovery of brain phosphorus metabolites and lactic acid level during reperfusion after cerebral ischemia in the rat – study by 13P and 1HNMR spectroscopy, Brain Res., 643 (1994) 92–99. [4] Barks, J.D.E. and Silverstein, F.S., Excitatory amino acids contribute to the pathogenesis of perinatal hypoxic-ischemic injury, Brain Pathol., 2 (1992) 235–243. [5] Blokland, A., Raaijmakers, W., van der Staay, J.F. and Jolles, J., Differential effect of acetyl-l-carnitine on open field behavior in young and old rats, Physiol. Behav., 47 (1990) 783–785. [6] Curti, D., Dagani, F., Galmozzi, M.R. and Marzatico, F., Effect of aging and acetyl-l-carnitine on energetic and cholinergic metabolism in rat brain regions, Mech. Aging Dev., 47 (1989) 39–45. [7] Dell’Anna, M.E., Calzolari, S., Molinari, M., Iuvone, L. and Calimici, R., Neonatal anoxia induces transitory hyperactivity, permanent spatial memory deficits and CA1 cell density reduction in developing rats, Behav. Brain Res., 45 (1991) 125–134. [8] Dell’Anna, M.E., Luthman, J., Lindqvist, E. and Olson, L., Development of monoamine systems after neonatal anoxia in rats, Brain Res. Bull., 32 (1993) 159–170. [9] Dell’Anna, M.E., Geloso, M.C., Draisci, G. and Luthman, J., Transient changes in fos and GFAp immunoreactivity precede neuronal cell loss in the rat hippocampus following neonatal anoxia, Exp. Neurol., 131 (1995) 144–156. [10] Dell’Anna, E., Geloso, M.C., Magarelli, M. and Molinari, M., Development of gamma-aminobutyric acid (GABA) and calcium binding proteins immunoreactivity in the rat hippocampus following neonatal anoxia, Neurosci. Lett., 211 (1996) 93–96. [11] Filloux, F.M., Adair, J. and Narang, N., The temporal evolution of striatal dopamine receptor binding and mRNA expression following hypoxia-ischemia in the neonatal rat, Dev. Brain Res., 94 (1996) 81–91. [12] Forloni, G., Angeretti, N. and Smiroldo, S., Neuroprotective activity of acetyl-l-carnitine: studies in vitro, J. Neurosci. Res., 37 (1994) 92–96. [13] Hershkowitz, M., Grimm, V. and Speiser, Z., The effects of postnatal anoxia on behavior and on muscarinic and beta-adrenergic receptors in the hippocampus of the developing rats, Dev. Brain Res., 7 (1983) 147–155. [14] Hill, A. and Volpe, J.J., Seizures, hypoxic-ischemic injury and intraventricular hemorrhage in the newborn, Ann. Neurol., 10 (1981) 109–121. [15] Imperato, A., Ramacci, M.T. and Angelucci, L., Acetyl-l-carnitine
E. Dell’Anna et al. / Neuroscience Letters 223 (1997) 201–205
[16]
[17]
[18]
[19]
[20]
enhances acetylcholine release in the striatum and hippocampus of awake freely moving rats, Neurosci. Lett., 107 (1989) 251–255. Kohjimoto, Y., Ogawa, T., Matsumoto, M., Shirakawa, K., Kuwaki, T., Yasuda, H., Anami, K., Fuji, T., Satoh, H. and Ono, T., Effects of acetyl-l-carnitine on the brain lipofuscin content and emotional behavior in aged rats, Jpn. J. Pharmacol., 48 (1988) 365– 371. Lipton, S.A. and Kater, S.B., Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, Trends Neurosci., 12 (1989) 265–270. Morris, A.J. and Carey, E.M., Postnatal changes in the concentration of carnitine and acetylcarnitine in the rat brain, Dev. Brain Res., 8 (1983) 381–384. Pascale, A., Milano, S., Corsico, N., Lucchi, L., Battaini, F., Arrigoni Martelli, E., Trabucchi, M. and Govoni, S., Protein kinase C activation and antiamnesic effect of acetyl-l-carnitine: in vitro and in vivo studies, Eur. J. Pharmacol., 265 (1994) 1–7. Pettegrew, J.W., Klunk, W.E., Kanagasabai, P., Kanfler, J.N. and McClure, R.J., Clinical and neurochemical effects of acetyl-l-carnitine in Alzheimer’s disease, Neurobiol. Aging, 16 (1995) 1–4.
205
[21] Shaffer, S.Q., Shaffer, D., O’Connor, P.A. and Stockman, C.J., Hard thought on neurological ‘soft signs’. In M. Rutter (Ed.), Developmental Neuropsychiatry, Guilford Press, New York, 1983, pp. 133–143. [22] Siliprandi, N., Siliprandi, D. and Ciman, M., Stimulation of oxidation of mitochondrial fatty acids and of acetate by acetylcarnitine, Biochem. J., 96 (1965) 777–782. [23] Spagnoli, A., Lucca, U., Menasce, G., Bandera, L., Cizza, G., Forloni, G. and Tettamanti, M. et al., Long-term acetyl-l-carnitine treatment in Alzheimer’s disease, Neurology, 41 (1991) 1726– 1732. [24] Szatkowski, M. and Attwell, D., Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms, Trends Neurosci., 17 (1994) 359–365. [25] Valerio, C., Clementi, G., Spadaro, F., D’Agata, V., Raffaele, R., Grassi, M., Lauria, N. and Drago, F., The effects of acetyl-l-carnitine on experimental models of learning and memory deficits in the old rat, Funct. Neurol., 4 (1989) 387–390.