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Combining exercise and cyclooxygenase-2 inhibition does not ameliorate learning deficits after brain insult, despite an increase in BDNF levels
Brain Research 1046 (2005) 224 – 229 www.elsevier.com/locate/brainres
Short communication
Combining exercise and cyclooxygenase-2 inhibition does not ameliorate learning deficits after brain insult, despite an increase in BDNF levels O.L. Gobbo, S.M. O’Mara* Department of Psychology and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland Accepted 29 March 2005 Available online 10 May 2005
Abstract Neurodegeneration can produce behavioral impairments. Previously, we have found that inhibition of cyclooxygenase-2 activity or physical activity was neuroprotective during kainic-acid-induced neural loss. Here, we investigated the combined effect of exercise pre-insult and cyclooxygenase inhibitor treatment post-kainate-induced brain damage. However, in spite of an increase in BDNF levels, the combination did not improve behavioral performance in Morris watermaze and object exploration tasks. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration, neurotrophic factor: biological effects, neural basis of behavior, learning and memory: systems and functions—animals Topic: Disorders of the nervous system, neurotoxicity Keywords: Running; Celecoxib; Watermaze; Object displacement
Limbic seizures in the rat, induced by intracerebral or systemic injection of kainic acid, represent a valuable animal model for temporal lobe neurodegeneration, closely reflecting the clinical and neuropathological symptoms of the disorder in humans [1]. Indeed, the overstimulation of excitatory amino acid receptors by glutamate has been suggested as a major cause of injury or death of neurons [15] and has been implicated in brain insults such as stroke, hypoglycemia, trauma brain injury [18], epilepsy, as well as chronic neurodegenerative states such as Huntington’s disease, amyotrophic lateral sclerosis, and perhaps Alzheimer’s disease [6,16]. Kainic acid mediates damage through inflammatory pathways, causing an increase of prostaglandins [13] and free radicals [4]. Previous experiments have shown that selective cyclooxygenase-2 inhibitors (e.g., celecoxib) limited kainate-induced brain damage and functional/behavioral deficits [3,11] by inhibiting biosynthesis of prostaglandins [24]. Physical exercise may be another
* Corresponding author. Fax: +353 1 6712006. E-mail address: [email protected] (S.M. O’Mara). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.03.046
means to delay progression of brain insult. Animal studies have found that exercise leads to an attenuation of learning deficits following traumatic brain injury [12], kainateinduced neurodegeneration [10] or ischemia [22]. Exercise induces many changes in gene expression in the hippocampus [17,23]. We have focused this study on the possibility that the combination of both treatments should provide better protection against kainate-induced damage than each treatment separately. Experiments were carried out in strict adherence to national laws and international recommendations for the use and care of experimental animals (European Communities Council Directive of 24/11/86; 86/609/EEC). Male Wistar rats (250 –300 g) were submitted to an exercise regimen consisting of 5 days training in a running wheel overnight [21]. Rats voluntarily ran on average 500 m/day for 5 days, and the non-running group rats were placed in a nonmoving running wheel for similar periods of time in order to control for the effect of exposure to the apparatus. On the last day of training and post-running, the animals received a single injection of kainic acid (KA) intraperitoneally (i.p.; 12 mg/kg, Sigma-Aldrich, Dublin 24, Ireland) or equivalent
O.L. Gobbo, S.M. O’Mara / Brain Research 1046 (2005) 224 – 229
volume of its vehicle (saline). Animals were injected with celecoxib once or chronically (5 days; 6 or 40 mg/kg i.p., Celebrex, SEARLE division of Monsanto Ireland Ltd.), or its vehicle (10% DMSO) 2 h post-KA-treatment [11]. Three days later, the rats were trained to find the hidden platform submerged 2 cm below the water surface. The watermaze was a black circular pool filled with water at room temperature (diameter = 200 cm, height = 35 cm) placed in a room with many extra-maze cues. At the start of all trials (position randomized), the rat was placed in the water and swam until it found the platform where it waited for 15 s before removal [7]. The results were analyzed using twoway ANOVA, followed by Tukey’s post hoc test (P < 0.05). On examination of the performance of celecoxib-treated animals with the dose 6 mg/kg (+ run + KA, Fig. 1), a twoway ANOVA indicated an overall significant difference across day [time: F(7,1313) = 23.383, P < 0.001; distance: F(7,1264) = 7.045, P < 0.001], group [time: F(3,1313) = 318.569, P < 0.001; distance: F(3,1264) = 199.889, P < 0.001] and a significant interaction between day and group
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[time: F(21,1313) = 4.221, P < 0.001; distance: F(21,1264) = 3.234, P < 0.01]. KA groups (Run + KA + celecoxib 6 mg/ kg, 5 days; Run + KA + celecoxib 6 mg/kg once and KA + vehicle celecoxib) did not show a reduction in distance swam (or escape latency) to find the platform over days, whereas the control group (Run + vehicle KA + celecoxib, P < 0.01) improved over days of testing. The control group swam less than all other KA groups (P < 0.05) and also found the hidden platform faster than KA groups (P < 0.05). When the dose of celecoxib was increased to 40 mg/kg (+ run + KA, Fig. 2), a two-way ANOVA indicated an overall significant difference across day [time: F(7,1005) = 30.952, P < 0.001; distance: F(7,1005) = 17.477, P < 0.001], group (time: F(4,1005) = 117.538, P < 0.001; distance: F(4,1005) = 92.730, P < 0.001], and a significant interaction between day and group [time: F(28,1005) = 1.580, P < 0.001; distance: NS]. However, KA, Run + KA + celecoxib 40 mg/kg chronic, and Run + KA + celecoxib 40 mg/kg once groups did not reduce their swim distance to find the platform across days, the control [ F(7,279) = 20.328, P <
Fig. 1. There were no differences in escape latency and in distance swam between the KA (12 mg/kg) group and the Run + KA (12 mg/kg) + Cxb (celecoxib, 6 mg/kg) groups. However, controls (run + Vehicle + Cxb, 6 mg/kg) improved their performance over days (*P < 0.05, **P < 0.01, ***P < 0.001 versus KA group). n = 6 – 8 animals per treatment group.
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Fig. 2. There were no differences in escape latency and in distance swam between the KA (12 mg/kg) group and the Run + KA (12 mg/kg) + Cxb (celecoxib, 40 mg/kg) chronic or once groups. However, control (run + Vehicle + Cxb 40 mg/kg) and celecoxib 40 mg/kg groups learned better the platform position than the KA group (*P < 0.05, **P < 0.01, ***P < 0.001 versus KA group). n = 6 – 7 animals per treatment group.
0.001] and celecoxib 40 mg/kg [ F(7,239) = 16.963, P < 0.001] group improved over day of testing (escape latency and distance swam). After the watermaze task, animals were allowed rest for 2 days, before being tested in an object exploration task; the aim of this experiment was to test if there was any recovery of function in an alternative (and non-aversively motivated) mnemonic task. The rats were tested in the presence of four different objects over 6 sessions. On the first trial, each rat was allowed to explore the apparatus for 6 min. In the second trial, 4 objects (plastic jerry-can, pitcher, polystyrene box, and cement cylinder) were presented in the center of the apparatus and the rat was given 5 trials of 6 min duration with a 3-min intertrial interval (ITI). From trial 1 to trial 3, the 4 objects remained in the same place. In trial 4, the cement cylinder was substituted for the pitcher, which itself was moved to a new location (moved objects) at the periphery of the apparatus. Trial 5 was the same as trial 4.
As reported in previous experiments [11], normal rats are able to discriminate between familiar and new environments at the fourth trial, increasing exploration time of the moved objects, in contrast to rats with hippocampal damage. The basic measure was the time taken by the animals in exploring objects in different trials [11]. Fig. 3 illustrates the mean time of contacts made with (specific) moved objects. Upon examining the performance of celecoxib-treated animals with 6 mg/kg dose (Fig. 3A), a two-way ANOVA indicated an overall significant difference across trial [ F(4,110) = 6.485, P < 0.001], but there were no significant differences between group, nor an interaction between trial and group. A Kruskal – Wallis one-way ANOVA showed that there was a significant difference between trials for the control (vehicle KA + vehicle Cxb: v 2 = 11.328, df = 4, P = 0.023) and run + vehicle KA + cxb 6 mg/kg (v 2 = 13.374, df = 4, P < 0.01). Post hoc Mann – Whitney test showed a significant
O.L. Gobbo, S.M. O’Mara / Brain Research 1046 (2005) 224 – 229
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Fig. 3. Object exploration task: Schematic representation of series of six consecutive exploratory trials in the open field arena. Control (= vehicle KA + vehicle celecoxib and vehicle KA + Cxb 6 mg/kg) and Run + vehicle KA + Cxb (celecoxib, 6 mg/kg) groups (panel A) and Control (= vehicle KA + vehicle celecoxib and RUN + veh KA + Cxb 40 mg/kg groups) and celecoxib, 40 mg/kg groups (panel B) groups decreased exploration over trials and demonstrated a significant difference between trial 3 (habituation) and trial (spatial). Mann – Whitney test, *P < 0.05.
difference between trial 3 (habituation) and trial 4 (reaction to spatial changes) for control and run + vehicle KA + cxb 6 mg/kg groups (P < 0.05). When the dose of celecoxib was increased to 40 mg/kg (Fig. 3B), a two-way ANOVA indicated an overall significant difference across trial [ F(4,100) = 4.044, P < 0.01] and between groups [ F(4,100) = 2.497, P < 0.05], but there was no significant interaction between trial and group. A Kruskal –Wallis one-way ANOVA showed that there were significant differences between trials for the control (vehicle KA + vehicle Cxb and run + vehicle KA + Cxb 40mg/kg: v 2 = 11.328, df = 4, P = 0.023); Run + KA + celecoxib 5 days (v 2 = 12.151, df = 4, P = 0.016); and celecoxib 40 mg/kg (v 2 = 7.972, df = 4, P = 0.033) groups. Post hoc tests showed a significant difference between trial 3 (habituation) and trial 4 (reaction to spatial changes) only for control and the celecoxib 40 mg/kg groups (P < 0.05).
At the end of the experiment, all animals were killed, their brain removed and dentate gyrus was taken from one hemisphere for measurement of BDNF levels (Fig. 4, BDNF ELISA procedure, [11, 21]). A one-way ANOVA indicated that there were significant differences between the groups [ F(5,39) = 35.590, P < 0.001]. Post hoc Tukey tests indicated that KA, RUN + KA + Cxb 6 mg/kg and RUN + KA + Cxb 40 mg/kg groups had more BDNF protein than the control group (P < 0.01). The second hemisphere was used for histology. Systemic KA administration induced widespread hippocampal neuron destruction (in areas CA1, CA2, and CA3), with the same histological profile that we have previously published ([10]; data not shown). This study demonstrated that the combination of exercise + celecoxib (at either dose) applied to KA-seizure rats, abolished the positive effect of each treatment individually
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Fig. 4. The graph shows the quantity of BDNF in the dentate gyrus in the right hippocampus of rats at the end of the experiment. BDNF levels were not significantly different between RUN + KA + Cxb once and chronic groups, then they were merged into two groups RUN + KA + Cxb 6 mg/kg and RUN + KA + Cxb 40 mg/kg. Post hoc test Tukey, **P < 0.01, ***P < 0.001.
(celecoxib [11] and exercise [10]) in spatial watermaze and object exploration tasks. Indeed, in previous experiments, we found that both celecoxib and exercise (each in isolation) allowed KA-treated animals to improve their performance in spatial and non-spatial tasks [10,11] relative to untreated controls. Running increases numerous mRNAs, some of which are involved in plasticity, and particularly increases BDNF mRNA, which is more involved in neurotrophic and neuroprotective action [8]. This may explain the improvement in the different task performances after KA injection, which increases both BDNF and COX-2 mRNA [8]. BDNF increase permits recovery of cells and attenuates the effect of increased COX-2 and of PGE2, which enhance membrane excitability [5]. COX-2 inhibitors block the PGE2 production induced after KA injection, attenuating inflammation and increased membrane excitability, permitting improvements in the watermaze and object exploration tasks. We hypothesize that, in these two cases (running and cyclooxygenase-2 inhibitor), BDNF production plays a role in tissue repair, permitting viable cells to compensate for any tissue loss. Other experiments have shown that BDNF can under some conditions exacerbate the injury caused by KA [19]; Lahteinen et al. [14] showed that transgenic mice overexpressing truncated TrkB (a dominant-negative receptor of BDNF) had less severe KA-induced seizures with later onset and lower mortality; and Croll et al. [9] have found that transgenic mice overexpressing BDNF exhibit an increased severity of KA-induced seizure; BDNF can also induce hyper-excitability in the mossy fibers of an epileptic hippocampus [20]. In conclusion, we hypothesized that the combination of running pre-insult and celecoxib after KA-induced seizure together would improve the performance of the animals in the watermaze and object exploration tasks relative to KA-
treated animals. We found that this combination treatment did not, in fact, rescue performance relative to the KA only treated group, despite of an increase of BDNF levels. BDNF has been described as an important factor in synaptogenesis and has neuroprotective effects. However, it has been also implicated in epileptogenesis [2]. We suggest that, in our conditions, by a unclear mechanism, BDNF became harmful. This was probably due to a hyper-excitability, preventing the BDNF-induced formation of new synaptic contacts (sprouting) or inducing an inconsistent alteration of synaptic strengths [2].
Acknowledgments Supported by the Higher Education Authority Research Programme on Neurodegeneration and the Programme for Research in Third Level Institutions.
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[15] B.S. Meldrum, Glutamate as a neurotransmitter in the brain: review of physiology and pathology, J. Nutr. 130 (Suppl. 4S) (2000) 1007S – 1015S. [16] B. Meldrum, J. Garthwaite, Excitatory amino acid neurotoxicity and neurodegenerative disease, Trends Pharmacol Sci. 11 (9) (1990) 379 – 387. [17] R. Molteni, Z. Ying, F. Gomez-Pinilla, Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarray, Eur. J. Neurosci. 16 (6) (2002) 1107 – 1116. [18] O. Piot-Grosjean, F. Wahl, O. Gobbo, J.M. Stutzmann, Assessment of sensorimotor and cognitive deficits induced by a moderate traumatic injury in the right parietal cortex of the rat, Neurobiol. Dis. 8 (2001) 1082 – 1093. [19] J.S. Rudge, P.E. Mather, E.M. Pasnikowski, N. Cai, T. Corcoran, et al., Endogenous BDNF protein is increased in adult rat hippocampus after a kainic acid induced excitotoxic insult but exogenous BDNF is not neuroprotective, Exp. Neurol. 149 (2) (1998) 398 – 410. [20] H.E. Scharfman, J.H. Goodman, A.L. Sollas, Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus, J. Neurosci. 19 (13) (1999) 5619 – 5631. [21] K.N. Shaw, S. Commins, S.M. O’Mara, Deficits in spatial learning and synaptic plasticity induced by the rapid and competitive broadspectrum cyclooxygenase inhibitor ibuprofen are reversed by increasing endogenous brain-derived neurotrophic factor, Eur. J. Neurosci. 17 (11) (2003) 2438 – 2446. [22] W. Stummer, A. Baethmann, R. Murr, L. Schurer, O.S. Kempski, Cerebral protection against ischemia by locomotor activity in gerbils. Underlying mechanisms, Stroke 26 (8) (1995) 1423 – 1429 (discussion 1430). [23] L. Tong, H. Shen, V.M. Perreau, R. Balazs, C.W. Cotman, Effects of exercise on gene-expression profile in the rat hippocampus, Neurobiol. Dis. 8 (6) (2001) 1046 – 1056. [24] H. Vanegas, H.G. Schaible, Prostaglandins and cycloxygenases in the spinal cord, Prog. Neurobiol. 64 (4) (2001) 327 – 363.
Short communication
Combining exercise and cyclooxygenase-2 inhibition does not ameliorate learning deficits after brain insult, despite an increase in BDNF levels O.L. Gobbo, S.M. O’Mara* Department of Psychology and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland Accepted 29 March 2005 Available online 10 May 2005
Abstract Neurodegeneration can produce behavioral impairments. Previously, we have found that inhibition of cyclooxygenase-2 activity or physical activity was neuroprotective during kainic-acid-induced neural loss. Here, we investigated the combined effect of exercise pre-insult and cyclooxygenase inhibitor treatment post-kainate-induced brain damage. However, in spite of an increase in BDNF levels, the combination did not improve behavioral performance in Morris watermaze and object exploration tasks. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration, neurotrophic factor: biological effects, neural basis of behavior, learning and memory: systems and functions—animals Topic: Disorders of the nervous system, neurotoxicity Keywords: Running; Celecoxib; Watermaze; Object displacement
Limbic seizures in the rat, induced by intracerebral or systemic injection of kainic acid, represent a valuable animal model for temporal lobe neurodegeneration, closely reflecting the clinical and neuropathological symptoms of the disorder in humans [1]. Indeed, the overstimulation of excitatory amino acid receptors by glutamate has been suggested as a major cause of injury or death of neurons [15] and has been implicated in brain insults such as stroke, hypoglycemia, trauma brain injury [18], epilepsy, as well as chronic neurodegenerative states such as Huntington’s disease, amyotrophic lateral sclerosis, and perhaps Alzheimer’s disease [6,16]. Kainic acid mediates damage through inflammatory pathways, causing an increase of prostaglandins [13] and free radicals [4]. Previous experiments have shown that selective cyclooxygenase-2 inhibitors (e.g., celecoxib) limited kainate-induced brain damage and functional/behavioral deficits [3,11] by inhibiting biosynthesis of prostaglandins [24]. Physical exercise may be another
* Corresponding author. Fax: +353 1 6712006. E-mail address: [email protected] (S.M. O’Mara). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.03.046
means to delay progression of brain insult. Animal studies have found that exercise leads to an attenuation of learning deficits following traumatic brain injury [12], kainateinduced neurodegeneration [10] or ischemia [22]. Exercise induces many changes in gene expression in the hippocampus [17,23]. We have focused this study on the possibility that the combination of both treatments should provide better protection against kainate-induced damage than each treatment separately. Experiments were carried out in strict adherence to national laws and international recommendations for the use and care of experimental animals (European Communities Council Directive of 24/11/86; 86/609/EEC). Male Wistar rats (250 –300 g) were submitted to an exercise regimen consisting of 5 days training in a running wheel overnight [21]. Rats voluntarily ran on average 500 m/day for 5 days, and the non-running group rats were placed in a nonmoving running wheel for similar periods of time in order to control for the effect of exposure to the apparatus. On the last day of training and post-running, the animals received a single injection of kainic acid (KA) intraperitoneally (i.p.; 12 mg/kg, Sigma-Aldrich, Dublin 24, Ireland) or equivalent
O.L. Gobbo, S.M. O’Mara / Brain Research 1046 (2005) 224 – 229
volume of its vehicle (saline). Animals were injected with celecoxib once or chronically (5 days; 6 or 40 mg/kg i.p., Celebrex, SEARLE division of Monsanto Ireland Ltd.), or its vehicle (10% DMSO) 2 h post-KA-treatment [11]. Three days later, the rats were trained to find the hidden platform submerged 2 cm below the water surface. The watermaze was a black circular pool filled with water at room temperature (diameter = 200 cm, height = 35 cm) placed in a room with many extra-maze cues. At the start of all trials (position randomized), the rat was placed in the water and swam until it found the platform where it waited for 15 s before removal [7]. The results were analyzed using twoway ANOVA, followed by Tukey’s post hoc test (P < 0.05). On examination of the performance of celecoxib-treated animals with the dose 6 mg/kg (+ run + KA, Fig. 1), a twoway ANOVA indicated an overall significant difference across day [time: F(7,1313) = 23.383, P < 0.001; distance: F(7,1264) = 7.045, P < 0.001], group [time: F(3,1313) = 318.569, P < 0.001; distance: F(3,1264) = 199.889, P < 0.001] and a significant interaction between day and group
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[time: F(21,1313) = 4.221, P < 0.001; distance: F(21,1264) = 3.234, P < 0.01]. KA groups (Run + KA + celecoxib 6 mg/ kg, 5 days; Run + KA + celecoxib 6 mg/kg once and KA + vehicle celecoxib) did not show a reduction in distance swam (or escape latency) to find the platform over days, whereas the control group (Run + vehicle KA + celecoxib, P < 0.01) improved over days of testing. The control group swam less than all other KA groups (P < 0.05) and also found the hidden platform faster than KA groups (P < 0.05). When the dose of celecoxib was increased to 40 mg/kg (+ run + KA, Fig. 2), a two-way ANOVA indicated an overall significant difference across day [time: F(7,1005) = 30.952, P < 0.001; distance: F(7,1005) = 17.477, P < 0.001], group (time: F(4,1005) = 117.538, P < 0.001; distance: F(4,1005) = 92.730, P < 0.001], and a significant interaction between day and group [time: F(28,1005) = 1.580, P < 0.001; distance: NS]. However, KA, Run + KA + celecoxib 40 mg/kg chronic, and Run + KA + celecoxib 40 mg/kg once groups did not reduce their swim distance to find the platform across days, the control [ F(7,279) = 20.328, P <
Fig. 1. There were no differences in escape latency and in distance swam between the KA (12 mg/kg) group and the Run + KA (12 mg/kg) + Cxb (celecoxib, 6 mg/kg) groups. However, controls (run + Vehicle + Cxb, 6 mg/kg) improved their performance over days (*P < 0.05, **P < 0.01, ***P < 0.001 versus KA group). n = 6 – 8 animals per treatment group.
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Fig. 2. There were no differences in escape latency and in distance swam between the KA (12 mg/kg) group and the Run + KA (12 mg/kg) + Cxb (celecoxib, 40 mg/kg) chronic or once groups. However, control (run + Vehicle + Cxb 40 mg/kg) and celecoxib 40 mg/kg groups learned better the platform position than the KA group (*P < 0.05, **P < 0.01, ***P < 0.001 versus KA group). n = 6 – 7 animals per treatment group.
0.001] and celecoxib 40 mg/kg [ F(7,239) = 16.963, P < 0.001] group improved over day of testing (escape latency and distance swam). After the watermaze task, animals were allowed rest for 2 days, before being tested in an object exploration task; the aim of this experiment was to test if there was any recovery of function in an alternative (and non-aversively motivated) mnemonic task. The rats were tested in the presence of four different objects over 6 sessions. On the first trial, each rat was allowed to explore the apparatus for 6 min. In the second trial, 4 objects (plastic jerry-can, pitcher, polystyrene box, and cement cylinder) were presented in the center of the apparatus and the rat was given 5 trials of 6 min duration with a 3-min intertrial interval (ITI). From trial 1 to trial 3, the 4 objects remained in the same place. In trial 4, the cement cylinder was substituted for the pitcher, which itself was moved to a new location (moved objects) at the periphery of the apparatus. Trial 5 was the same as trial 4.
As reported in previous experiments [11], normal rats are able to discriminate between familiar and new environments at the fourth trial, increasing exploration time of the moved objects, in contrast to rats with hippocampal damage. The basic measure was the time taken by the animals in exploring objects in different trials [11]. Fig. 3 illustrates the mean time of contacts made with (specific) moved objects. Upon examining the performance of celecoxib-treated animals with 6 mg/kg dose (Fig. 3A), a two-way ANOVA indicated an overall significant difference across trial [ F(4,110) = 6.485, P < 0.001], but there were no significant differences between group, nor an interaction between trial and group. A Kruskal – Wallis one-way ANOVA showed that there was a significant difference between trials for the control (vehicle KA + vehicle Cxb: v 2 = 11.328, df = 4, P = 0.023) and run + vehicle KA + cxb 6 mg/kg (v 2 = 13.374, df = 4, P < 0.01). Post hoc Mann – Whitney test showed a significant
O.L. Gobbo, S.M. O’Mara / Brain Research 1046 (2005) 224 – 229
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Fig. 3. Object exploration task: Schematic representation of series of six consecutive exploratory trials in the open field arena. Control (= vehicle KA + vehicle celecoxib and vehicle KA + Cxb 6 mg/kg) and Run + vehicle KA + Cxb (celecoxib, 6 mg/kg) groups (panel A) and Control (= vehicle KA + vehicle celecoxib and RUN + veh KA + Cxb 40 mg/kg groups) and celecoxib, 40 mg/kg groups (panel B) groups decreased exploration over trials and demonstrated a significant difference between trial 3 (habituation) and trial (spatial). Mann – Whitney test, *P < 0.05.
difference between trial 3 (habituation) and trial 4 (reaction to spatial changes) for control and run + vehicle KA + cxb 6 mg/kg groups (P < 0.05). When the dose of celecoxib was increased to 40 mg/kg (Fig. 3B), a two-way ANOVA indicated an overall significant difference across trial [ F(4,100) = 4.044, P < 0.01] and between groups [ F(4,100) = 2.497, P < 0.05], but there was no significant interaction between trial and group. A Kruskal –Wallis one-way ANOVA showed that there were significant differences between trials for the control (vehicle KA + vehicle Cxb and run + vehicle KA + Cxb 40mg/kg: v 2 = 11.328, df = 4, P = 0.023); Run + KA + celecoxib 5 days (v 2 = 12.151, df = 4, P = 0.016); and celecoxib 40 mg/kg (v 2 = 7.972, df = 4, P = 0.033) groups. Post hoc tests showed a significant difference between trial 3 (habituation) and trial 4 (reaction to spatial changes) only for control and the celecoxib 40 mg/kg groups (P < 0.05).
At the end of the experiment, all animals were killed, their brain removed and dentate gyrus was taken from one hemisphere for measurement of BDNF levels (Fig. 4, BDNF ELISA procedure, [11, 21]). A one-way ANOVA indicated that there were significant differences between the groups [ F(5,39) = 35.590, P < 0.001]. Post hoc Tukey tests indicated that KA, RUN + KA + Cxb 6 mg/kg and RUN + KA + Cxb 40 mg/kg groups had more BDNF protein than the control group (P < 0.01). The second hemisphere was used for histology. Systemic KA administration induced widespread hippocampal neuron destruction (in areas CA1, CA2, and CA3), with the same histological profile that we have previously published ([10]; data not shown). This study demonstrated that the combination of exercise + celecoxib (at either dose) applied to KA-seizure rats, abolished the positive effect of each treatment individually
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Fig. 4. The graph shows the quantity of BDNF in the dentate gyrus in the right hippocampus of rats at the end of the experiment. BDNF levels were not significantly different between RUN + KA + Cxb once and chronic groups, then they were merged into two groups RUN + KA + Cxb 6 mg/kg and RUN + KA + Cxb 40 mg/kg. Post hoc test Tukey, **P < 0.01, ***P < 0.001.
(celecoxib [11] and exercise [10]) in spatial watermaze and object exploration tasks. Indeed, in previous experiments, we found that both celecoxib and exercise (each in isolation) allowed KA-treated animals to improve their performance in spatial and non-spatial tasks [10,11] relative to untreated controls. Running increases numerous mRNAs, some of which are involved in plasticity, and particularly increases BDNF mRNA, which is more involved in neurotrophic and neuroprotective action [8]. This may explain the improvement in the different task performances after KA injection, which increases both BDNF and COX-2 mRNA [8]. BDNF increase permits recovery of cells and attenuates the effect of increased COX-2 and of PGE2, which enhance membrane excitability [5]. COX-2 inhibitors block the PGE2 production induced after KA injection, attenuating inflammation and increased membrane excitability, permitting improvements in the watermaze and object exploration tasks. We hypothesize that, in these two cases (running and cyclooxygenase-2 inhibitor), BDNF production plays a role in tissue repair, permitting viable cells to compensate for any tissue loss. Other experiments have shown that BDNF can under some conditions exacerbate the injury caused by KA [19]; Lahteinen et al. [14] showed that transgenic mice overexpressing truncated TrkB (a dominant-negative receptor of BDNF) had less severe KA-induced seizures with later onset and lower mortality; and Croll et al. [9] have found that transgenic mice overexpressing BDNF exhibit an increased severity of KA-induced seizure; BDNF can also induce hyper-excitability in the mossy fibers of an epileptic hippocampus [20]. In conclusion, we hypothesized that the combination of running pre-insult and celecoxib after KA-induced seizure together would improve the performance of the animals in the watermaze and object exploration tasks relative to KA-
treated animals. We found that this combination treatment did not, in fact, rescue performance relative to the KA only treated group, despite of an increase of BDNF levels. BDNF has been described as an important factor in synaptogenesis and has neuroprotective effects. However, it has been also implicated in epileptogenesis [2]. We suggest that, in our conditions, by a unclear mechanism, BDNF became harmful. This was probably due to a hyper-excitability, preventing the BDNF-induced formation of new synaptic contacts (sprouting) or inducing an inconsistent alteration of synaptic strengths [2].
Acknowledgments Supported by the Higher Education Authority Research Programme on Neurodegeneration and the Programme for Research in Third Level Institutions.
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