ЭКСПЕРИМЕНТАЛЬНЫЕ РАБОТЫ
УДК 577
FORELIMB AKINESIA AND METABOLIC ALTERATION IN THE STRIATUM
FOLLOWING UNILATERAL 6-HYDROXYDOPAMINE LESION IN RATS: AN IN VIVO PROTON MAGNETIC RESONANCE SPECTROSCOPY STUDY
© 2011 S. Y. Kim", B. Y. Choe", H. S. Lee4, D. W. Lee", K. N. Ryuc, J. S. Parkc, C. S. Yind,
K. S. Hong4, C. H. Lee4, and C. B. Choie*
aDepartment of Biomedical Engineering and Research Institute of Biomedical Engineering, College of Medicine, The Catholic
University of Korea, Seoul, Republic of Korea bDivision of Magnetic Resonance Research, Korea Basic Science Institute, Choongbuk, Korea cDepartment of Radiology, Kyung Hee University Medical Center, Seoul, Korea dAcupuncture and Meridian Science Research Center, Kyung, Seoul, Republic of Korea eDepartment of Veterinary Diagnostic Radiology, Dr. PET Animal Medical Center, Seoul, Korea
Abstract—The 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopamine system in rat can develop neuropathological and neurochemcial changes similar to those seen in patients with Parkinson's disease (PD). The purpose of this study was to test the hypothesis that the N-acetylaspartate level (NAA), regarded as neuronal marker would be decreased after 6-OHDA lesions in rat brain and to determine whether metabolic alteration are correlated with behavioral deficit. The animals were undergone adjusting stepping test and proton magnetic resonance spectroscopy (XH-MRS). In vivo 'H-NMR spectra (TR/TE = 2500/144 ms) were acquired from both left (contralateral region) and right striatum (ipsillateral region). There was a highly significant impairment in left forepaw performance and significant reductions in NAA/total creatine (tCr) ratios were observed in the ipsillateral striatum of rat (P < 0.05). Furthermore, there was a significant correlation between left forepaw performance and NAA/tCr level on the whole sample (Spearman correlation test, p = 0.634, P = 0.011). Our results suggest that the NAA/tCr ratio may be a valuable criterion for evaluation of functional impairment of the striatum in 6-OHDA rat model of PD.
Keywords: Parkinson's disease (PD), 6-hydroxydopamine (6-OHDA), proton magnetic resonance spectroscopy (1H-MRS), striatum, N-acetylaspartate (NAA).
INTRODUCTION
Parkinson's disease (PD) is a neurological disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and concomitant loss of dopamine (DA) in the striatum [1]. Several pathogenic mechanisms have been discovered that contribute to neuronal cell loss in PD [2]. This loss continues to increase after diagnosis, despite the use of dopamineric and/or adrenergic drugs as therapy to maintain proper movement function for some period. The mechanism for continued neuron cell death in the substantia nigra is currently unknown.
Animal models can provide an important aid to investigate the pathogenic mechanisms and therapeutic strategies in human diseases. The 6-hydroxydopamine (6-OHDA) has become one of the most widely used neurotoxins for modeling PD in experimental animals. Rats with 6-OHDA-induced lesions of the nigrostriatal
* Corresponding author: Department of Veterinary Diagnostic Radiology, Dr. PET Animal Medical Center, 35-3 SamsungDong, Kangnam-Gu, Seoul, Korea, 135-867, Phone: 82-22258-7503, Fax: 82-2-2675-0010, e-mail: sgivet@gmail.com.
dopamine system develop neuropathological and neu-rochemcial changes similar to those seen in patients with PD [3]. Following 6-OHDA injections into SN or the nigrostriatal tract [4, 5], dopaminergic neurons start degenerating within 24 hours after, and striatal dopam-ine is depleted 2 to 3 days later [6]. Although 6-OHDA-induced lesions have been described in various species including zebrafish [7], guinea pig [8], mice [9, 10], and cats [11, 12], rats are most commonly used because of established stereotactic technique and relatively low maintenance costs. Through the use of an animal model, striatal dopamine deficiency was known to be associated with symptoms of PD.
The brain metabolite N-acetylaspartate (NAA), a reduction in which correlates with a loss of neuronal density or integrity can be measured by use of proton magnetic resonance spectroscopy (1H-MRS) technique. The localized in vivo 1H-MRS has recently become practical tools to assess brain metabolic changes in various neurological diseases and can provide neu-rochemical information (tCr, total creatine; tCho, total choline; Glu + Gln, glutamate+glutamine; Lac,
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lactate) about the progress of degenerative changes in the brain [13]. Abnormal metabolic ratios of the NAA have been observed in the posterior cingulate of PD patients using 1H-MRS [14]. In addition, recent 1H-MRS studies of rat model of PD showed that the ratio of NAA/Cr in the right frontal cortex was significantly lower in PD model than in the control group [15].
The purpose of this study was to test the hypothesis that the NAA level, regarded as neuronal marker would be decreased after 6-OHDA lesions in rat brain and to determine whether metabolic alteration are correlated with behavioral deficit. Two volume-of-in-terests (VOIs) were selected, one centered in the right and the other centered in the left striatum. The stria-tum was chosen because most nigrostriatal afferents terminate in this region. Based on previous PD research, we hypothesized that 6-OHDA lesion in rat brain would cause the reduction of the neuronal marker (i.e., NAA).
MATERIALS AND METHODS
Animals
Fifteen male Sprague-Dawley rats (SCL, Shizuoka, Japan), aged 6 weeks with a weight of 160—180 g at the start of the experiment were housed under controlled environmental conditions (temperature 23 ± 2°C, 12 : 12-h light-dark cycle). The animals were divided into two groups, one is 6-OHDA injected rat (N = 5) and the other is control rat treated with saline (N = 10). The animals were provided with food and water ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee (Konkuk University, Korea). The minimum number of animals required to analyze statistical differences was employed.
Unilateral 6-OHDA Injections
Before the unilateral lesions of the right striatum, the rats were anesthetized with 40 mg/kg sodium pen-tobarbital i.p., and were placed in a stereotaxic frame. The 6-OHDA (Sigma, Saint Louis, MO, USA) was dissolved at a concentration of 5 mg/ml in 0.9% saline containing 0.2% ascorbic acid (Sigma) to avoid 6-OHDA oxidation. A total dose of 25 ^g in 5 ^l was injected into the right medial forebrain bundle at a flow rate of 0.5 ^l/min, using a Hamilton microsy-ringe fitted with a 26-gauge cannula. Lesion coordinates were as follows: anterior-posterior (AP) — 4.8 mm, medial-lateral (ML) — 1.7 mm, dorsal-ventral (DV) — 8.4 mm from the dura [16]. The cannula was initially placed stereotactically at the coordinate position and left in the place for 1 min before starting the infusion. After the injection, the cannula remained in the place for an additional 5 min to allow diffusion of the toxin, before being slowly withdrawn. The control rats underwent through the same surgical procedure, but received 5 ul of saline with 0.2% ascorbic acid.
Adjusting Stepping Test
Four weeks after the 6-OHDA injection, the adjusting stepping test was conducted to evaluate the effects of 6-OHDA lesions on the forelimb function. Prior to the test, the rats were handled by experimenter to become familiar with the experimenter's grip for one week. In most studies, stepping movements are produced when the experimenter slowly moves the rat laterally and the number of steps produced per second is used to assess akinesia [17, 18]. As a modification in the present study, a moving treadmill belt was used to generate a stepping event while the experimenter and rat remain stationary, allowing for more consistency in the speed of movements between trials [19].
During the test, the rat was held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface and placed on the surface of the treadmill (Columbus Instruments, Columbus, OH USA) that was set to move at a rate of 70 cm/10 sec in the direction opposite to the weight-bearing forepaw. The belt of the treadmill was made of flexible rubber and the surface was covered with cloth tape to give a textured surface for the forepaw movements. The number of adjusting steps of the weight-bearing forepaw to compensate for the movement of the body was counted, as described previously [17]. Each stepping test consisted of three trials for each forepaw, alternating between each forepaw. In all experiments, the average of the three trials for each forepaw was used for the analysis. The control rats were also included in the stepping test as described above.
In vivo 1H-NMR Spectra Acquisitions
In vivo 1H-MRS measurements were performed following the adjusting stepping test. All MR experiments were conducted with a 4.7T BIOSPEC scanner (Bruker Medical, Ettlingen, Germany) with use of a 400 mm bore magnet and 150 mT/m actively shielded gradient coils. To execute MRS measurements, rats were initially anesthetized by inhalation of isoflurane at a 4—5% concentration in a 5:5 mixture of N2O and O2, and anesthesia was maintained by inhalation of a 1.5—2% concentration of isoflurane in a 5:5 mixture of N2O and O2. Anesthetized rats were placed in the prone position with the head firmly fixed on a palate holder equipped with an adjustable nose cone. A scout image was initially obtained to verify the position of the animal and the image quality. The position ofVOIs (contralateral and ipsillateral striatum, each volume: 2.5 x 2.5 x 2.5 mm3) was carefully selected from mul-tislice axial T2-weighted MR images obtained using rapid acquisition with a relaxation enhancement (RARE) sequence (TR/TE = 5000/22 ms, slice thickness = 1.0 mm, NEX = 2, matrix size = 256 x 192) (Figs. 1, 2). The proton spectra were obtained with use of a poi
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