A maioria dos modelos utilizados para estudar os déficits cognitivos e motores da DP é baseada em efeitos de administrações agudas de substâncias como 6- OHDA e MPTP. Contudo, esses dois modelos acarretaram em perdas específicas e imediatas de células do sistema nervoso (SN) além de um acentuado número de mortes de animais, não apresentando um processo neurodegenerativo progressivo (Ferro et al. 2005, Meredith et al. 2008).
Uma alternativa proposta nesse trabalho seria a utilização de um modelo farmacológico crônico, administrando-se repetidamente baixas doses de reserpina, que possibilitasse o aparecimento progressivo de sinais semelhantes aos sintomas encontrados na DP. Estudos prévios com administração aguda de reserpina têm mostrado alterações motoras (Skalisz et al. 2002, Aguiar Jr et al. 2009), mas também um comprometimento cognitivo que ocorreria independentemente do declínio motor, como quadros clínicos encontrados em humanos com DP (Carvalho et al. 2006, Fernandes et al. 2008). Além disso, nesses estudos anteriores com
40 animais, a administração aguda subcutânea de reserpina (em doses que não afetam a função motora) levou a alterações de memória que envolve contexto emocional enquanto as sem conotação emocional não foram afetadas. Outro fator importante a ser considerado é que além da dopamina, há evidências da participação de outros sistemas de neurotransmissão, como o GABA e o glutamato na gênese dos sintomas parkinsonianos, tanto em modelos animais quanto em humanos (Bezard et al, 1997; Bianchi et al, 2003; Bonsi et al, 2007; DeLong e Wichmann, 2007). Existe ainda o fator da alteração de mecanismos de neuroproteção a eventos oxidativos induzidos pela reserpina (Bilska et al. 2007, Spina & Cohen 1989), os quais corroboram os danos do estresse oxidativo em cérebro de pacientes com DP (Beal 2002), e em modelos farmacológicos como o MPTP (Obata 2002) e a 6-OHDA (Riobó et al. 2002). Contudo, a relação entre o estresse oxidativo, o envolvimento de sistemas de neurotransmissão não dopaminérgicos, fatores relacionados à degeneração progressiva e alterações motoras e cognitivas na DP ainda não está completamente esclarecida.
Tomados em conjunto, dados prévios sugerem que o efeito induzido pela reserpina sobe a memória e parâmetros motores de ratos pode ser uma abordagem adequada para o estudo dos sintomas cognitivos e motores da DP. Entretanto, até o momento, tais estudos foram realizados apenas com a administração aguda de reserpina. Assim sendo, propomos o estudo da administração repetida de baixas doses de reserpina como um modelo farmacológico de DP que possa abranger as características citadas acima, assemelhando aos sintomas observados em humanos afetados por esta patologia.
41 1.4. Objetivo geral
No presente trabalho, propomos desenvolver um possível modelo farmacológico em ratos que mimetize uma neurodegeneração progressiva semelhante às encontradas em pacientes com DP, através da administração repetida de baixas doses de reserpina.
1.4.1. Objetivos específicos
Avaliamos os efeitos da administração repetida de reserpina (em doses que causariam pouco ou nenhum comprometimento motor agudamente) sobre:
1. O desempenho de ratos em modelos comportamentais de memória ao longo do tratamento;
2. A atividade motora ao longo do tratamento;
3. Os níveis de GABA e glutamato em regiões cerebrais possivelmente relacionadas com o surgimento de déficits cognitivos ou sintomas motores ao final do tratamento;
4. Os níveis de peroxidação lipídica como indicativo de dano causado por processos oxidativos decorrentes da administração repetida de reserpina.
42
43 2.1. Experimento I: Artigo científico que será submetido ao periódico Psychology & Neuroscience
BEHAVIORAL AND NEUROCHEMICAL EFFECTS OF
REPEATED ADMINISTRATION OF LOW DOSES OF
RESERPINE: A PROGRESSIVE MODEL FOR THE STUDY OF
PARKINSON’S DISEASE?
Valéria S. Fernandes1, Anderson H.F.F. Leão1, Angela Maria Ribeiro2, Alessandra M. Ribeiro1, Regina H. Silva1,*
1Memory Studies Laboratory, Physiology Department, Federal University of Rio
Grande do Norte, Natal, Brazil
2Departamento de Bioquímica e Imunologia, Laboratório de Neurociência Comportamental e Molecular – LaNeC. Universidade Federal de Minas Gerais, Brazil
44 Abstract
Parkinson's Disease (PD) has been studied in models that attempt to mimic the neurophysiologic and behavioral changes found in the development of this disease. However, in general, these protocols induce an immediate severe motor impairment, similar to advanced stages of PD. The administration of reserpine (a monoamine depletor) in rodents has been considered a model for studying PD. In this study, repeated treatment with 0.1 and 0.2 mg/kg (but not 0.05 mg/kg) reserpine have induced progressive motor alterations in rats when compared with the vehicle-treated group, as shown by the evaluation of the catalepsy behavior across the treatment. Additionaly, animals repeatedly treated with 0.1 mg/kg reserpine showed concomitant memory impairment when tested in the plus-maze discriminative avoidance task. At the end of the treatment (5 days after the 15th injection) striatal GABA and gluatamate levels were determined. While no changes were observed for the GABAergic system, a decrease in glutamate striatal concentration was found in 0.1 and 0.2 mg/kg reserpine-treated animals. Thus, repeated treatment with low doses of reserpine appears to be promising as a model of PD, since it induces progressive motor alterations. By the end of the treatment, these motor symptoms were accompanied by cognitive and neurochemical changes. However, more studies are needed to verify if the memory deficits and neurochemical alterations would present a progressive profile as well.
Keywords:
45 1. Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disease in which the ability to perform voluntary movements is gradually lost. The clinical condition of PD includes rigidity, tremor and bradykinesia (slowness of movement) (Klockgether, 2004). However, cognitive changes can also be observed in patients with PD (Aarsland et al., 2004; Mahieux et al., 1998; Verbaan et al., 2007).
Animal models have been used to study the changes caused by PD. Some studies try to mimic the clinical features of PD in rodents in order to better understand the neurophysiological mechanisms underlying the disorder (Meredith et al. 2008). However, the the effectiveness of models regarding the progressive nature of the "preclinical" and "clinical" stages of PD is controversial (Deumens et al., 2002). Indeed, in recent decades some pharmacological animal models of PD have been developed, with the most studied toxins being 6-hydroxydopamine (6-OHDA) and 1- methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) (Meredith et al., 2008; Schober, 2004). These two models have shown specific loss of cells related to PD in the central nervous system (CNS), although not presenting a neurodegenerative process, instating already an advanced stage of the disease upon administration (Meredith et al., 2008).
The administration of reserpine (an irreversible blocker of monoamine vesicular carrier) in rodents has been considered an animal model for the study of PD (Colpaert, 1987; Kim et al., 1999; Alves et al., 2000; Silva et al., 2002; Skalisy et al., 2002). Reserpine interferes with the storage of monoamines in intracellular vesicles, causing monoamine depletion in nerve terminals and transient hypolocomotion and muscular rigidity, depending on the dose (Colpaert, 1987).
46 Recently, lower doses of reserpine have also been found to promote a memory deficit in an aversive discrimination task without any effects on motor activity, suggesting that the administration of this drug in low doses can be useful to study memory deficits found in PD (Carvalho et al., 2006). Similar results were found in a different aversively motivated behavioral model, the contextual fear conditioning (Fernandes et al., 2008). In summary, the data from the literature suggest that acute reserpine is able to induce motor and cognitive alterations similar to those found in PD patients, although in different dose ranges.
Although hypofunction of the dopaminergic nigrostriatal system is considered to be the core of the physiopathology of PD, there is evidence that other neurotransmitter systems are involved in the symptoms of the disease (Bezard et al., 1997; Bianchi et al., 2003; Bonsi et al., 2007; DeLong & Wichmann, 2007). Studies have suggested that the depletion of dopamine (DA) in the striatum consequently leads to inhibition of the GABAergic striato-pallidal projections, as well as changes in thalamo-nigral glutamatergic projections (Filloux & Townsend, 1993; DeLong & Wichmann, 2007). Indeed, a study with rats treated with 6-OHDA showed that loss of dopaminergic neurons in the forebrain induce increase on the GABA levels in pallidum globe (Bianchi et al., 2003). Furthermore, studies with glutamatergic drugs (in particular, group II mGluR agonists) show improvement of motor signs in mice treated with MPTP (Bonsi et al., 2007). However, these studies in animals were performed with acute pharmacological models, and have shown controversial results. Further, studies with brain tissue from PD patients show altered levels of GABA in the medial center thalamus (Gerlach et al., 1996). These studies indicate that the relationship between dopaminergic, glutamatergic, GABAergic and behavioral changes is somewhat complex, and still unclear.
47 Considering the importance of an animal model that simulates the progressive nature of the disease, we evaluated the effects of a repeated treatment with low sub- effective doses of reserpine on motor and cognitive behaviors. In addition, we also addressed possible changes in GABAergic and glutamatergic systems as a consequence of this treatment.
2. Methods
2.1. Subjects
Five-month old male Wistar rats (n= 29) were used. All animals were maintained in groups of four or five per cage, under a 12 h light 12 h dark cycle and at a constant temperature of 25 1 C, with food and water available ad libitum. The rats were handled according to Brazilian law procedures for the use of animals in scientific research (Law Number 11.794) and all procedures were approved by the local research ethics committee (final opinion number 149/2008). All efforts were made to minimize animal pain, suffering or discomfort, and to minimize the number of rats used.
2.2. Drug treatment, general procedures and experimental design
Reserpine (methyl reserpate 3,4,5-trimetothoxycinnamic acid ester: Sigma Chemical Co. St. Louis, MO) was dissolved in glacial acetic acid and diluted to the correct concentration in distilled water. Vehicle consisted of the same amount of
48 acetic acid and water as in the reserpine solution. These solutions were injected subcutaneously (s.c.).
Rats received 15 s.c. injections of vehicle (VEH; n=8), 0.05 mg/Kg (RES 0.05; n=7), 0.1 mg/Kg (RES 0.1; n=7) or 0.2 mg/kg (RES 0.2; n=7) of reserpine, at a volume of 1 ml/kg body weight, on alternate days. During treatment rats went through the following procedures: (1) assessment of catalepsy behavior 24h after the 3rd, 6th, 9th, 12th and 15th injections; (2) plus-maze discriminative avoidance task 24h and 48 h after the 10th injection; (3) assessment of orofacial movements 24 h after the 14th injection; (4) Contextual fear conditioning 48 and 72 h after the 15th injection; (5) evaluation of GABAergic and glutamatergic parameters in the striatum 5 days after the 15th injection (Figure 1).
Figure 1: Experimental design
Every rat was submitted to 10 min of gentle handling once a day for five days before the beginning of the experimental procedures. The analyses of catalepsy
49 behavior and orofacial movements were performed by direct observation (by researchers blind to the treatment). All other behavioral sessions were recorded by a camera placed above the apparatus and the behavioral parameters were registered by an animal video-tracking software (Any maze Stoelting, USA).
All apparatus were washed with a water–alcohol (5%) solution before behavioral testing to eliminate possible bias due to odors left by previous subjects.
2.3. Apparatus
2.3.1. Catalepsy Test:
The catalepsy was assessed placing the animal‘s front paws on a horizontal bar positioned at 9 cm above the bench surface. The duration of catalepsy, which was defined as an immobile posture, keeping the two front paws on the bar, was measured within a maximum of 180 s.
2.3.2. Plus-maze discriminative avoidance task:
The apparatus employed was a modified elevated plus-maze made of wood containing two enclosed arms (50 X 15 X 40 cm) opposite to two open arms (50 X 15 cm). A 100-watt lamp was placed over the middle of one of the enclosed arms (aversive enclosed arm). In the training session, each rat was placed in the centre of the apparatus and, over a period of 10 min, every time the animal entered the enclosed arm containing the lamp, an aversive situation was produced until the animal left the arm. The aversive stimuli were the 100-watt light and an 80 dB noise
50 applied through a speaker placed over the aversive enclosed arm. In the test session, held 24h later, the rats were again placed in the apparatus for 10 min, without receiving the aversive stimulation, with the lamp and the speaker still present over the aversive arm, but turned off. Distance traveled in the apparatus (used for motor activity evaluation) and time spent in each arm (aversive, non-aversive and open arms) were registered. Percent time in aversive arm (time spent in aversive enclosed arm/time spent in both enclosed arms) and percent time spent in open arms (time spent in open arms/time spent in both open and enclosed arms) considering the whole duration of behavioral sessions were used to evaluate memory and anxiety, respectively (Silva et al., 2000). Percent time spent in the aversive enclosed arm assessed minute by minute across the training and test sessions were used to evaluate learning and extinction of the task, respectively (Ribeiro et al., 2010).
2.3.3. Orofacial movements assessment:
Rats were placed individually in wired cages (29 cm × 24 cm × 21 cm) with mirrors positioned under the floor and behind the back wall of the cage to allow behavioral quantification when the animal faced away from the observer. The number of tongue protrusions (projection of the tongue out of the oral cavity), vacuous chewing movement frequency (mouth openings in the vertical plane not directed toward physical material), and facial twitching (duration (in seconds) of twitching of the facial musculature) were measured continuously for 15 min.
51 2.4. Biochemical analysis: Evaluation of GABA and glutamate levels:
After the animals were sacrificed by decapitation, the brains were quickly removed from the cranial cavity, weighed and dissected according to the stereotactic coordinates provided by Paxinos & Watson (Paxinos & Watson 1997). The sample of striatum was then stored at -80 ° C to achieve the biochemical assays.
Samples of striatum were weighed and homogenized in 15 volumes of methanol: water (85:15 v / v) in automatic homogenizing. Then the homogenate was centrifuged at 4 ° C for 15 minutes at 7800x g (Sorvall RC-5B). The supernatant obtained after centrifugation was collected and kept on ice until subjected to derivatization.
Due to the absence of electroactive or fluorescent characteristics inherent in the amino acid glutamate and GABA, several works have used the technique of pre- column derivatization for the chromatographic separation and identification of these compounds. One of the most widely used derivatising agents is o-phthalaldehyde (OPA), which reacts with primary amines in the presence of thiol and generates electroactive and fluorescent derivatives (Freitas et al. 2009). The derivatization reaction was made by mixing 100 mL of sample, 20 mL of methanolic OPA (5 mg / mL) prepared daily, 75 mL of borate buffer (pH 9.9) and 5 mL of 3- mercaptopropiônico acid (MPA). The resulting solution was stirred and injected into the chromatographic system after 1 minute at room temperature.
The chromatographic system used to determine of GABA and glutamate consisted of a Shimadzu chromatograph (LC-10AD, Tokyo, Japan) with 200 mL injector valve (Rheodyne 7725-I, California, USA) and fluorescence detector (FLD- Shimadzu spectrofluorometric detector RF-551, Tokyo, Japan) coupled to a pump
52 LC-10. The wavelengths of excitation and emission used were 337 nm and 454 nm, respectively. A reversed phase chromatographic column C18 (150 mm x 4.6 mm ID) and a guard column (E. Merck RT 250-4, ER Darmsdt, Germany) were used in the analysis. The isocratic mobile phase consisted of a 0.05 M solution of sodium acetate, tetrahydrofuran and methanol (50:1:49 v / v), pH 4.0. The mean elution of GABA and glutamate is 8.0 to 3.0 minutes, respectively, the concentrations in µg / g of tissue were calculated using peak areas and their standard curves which was provided by an integrator (R7Ae Shimadzu C-plus) coupled to the chromatographic system (Freitas et al., 2009).
2.5. Statistics
All data were tested for homogeneity of variances (Levene's test) and normality (Kolmogorov-Smirnov test) and parametric tests were performed for all data. Data on the percentage of time spent in the aversive arm (measured every minute, in training and test sessions) and catalepsy behavior across the treatment (24h after the 3rd, 6th, 9th, 12th and 15th injections) were analyzed by analysis of variance (ANOVA) with repeated measures. For catalepsy behavior analysis, between-subject comparisons were held in each timepoint with one-way ANOVA with sequential Bonferroni‘s post hoc. Other data were analyzed by one-way ANOVA followed by Duncan's test for post hoc analysis. The results were expressed as means ± SE. The level of significance in all tests was p <0.05.
53 3. Results
3.1- Effects of repeated administration of low doses of reserpine on the catalepsy behavior:
Catelpsy behavior results are shown in figure 2. ANOVA with repeated measures revealed time (quantity of injections) [F(5, 125)=22.36; p=0.000],
treatment (reserpine (0.05, 0.1 or 0.2 mg/Kg) or vehicle) [F(3, 25)=16.92; p=0.000]
and time X treatment interaction [F(5, 125)=7.54; p=0.000] effects. Analyzing the data of each observation, we found no significant effects of treatment until the observation held after the 6th injection, although treatment with 0,2 mg/kg reserpine induced a marginally significant effect after six injections (p=0.068 compared to vehicle). Significant effects of treatment were observed after the 9th, 12th and 15th subcutaneous injections [F (3, 25) = 8.32, p = 0.001; F (3, 25) = 11.22, p = 0.000; F (3, 25) = 8.28, p = 0.000, respectively]. The Bonferroni‘s post hoc analysis showed that RES 0.1 and RES 0.2 groups presented increased immobility time in the bar when compared to the VEH and RES 0.05 groups after the 9th, 12th and 15th injections.
54 Fig. 2: Effects of repeated administration of low doses of reserpine (RES- 0.05, 0.1 or 0.2 mg/Kg) or vehicle (VEH) on catalepsy behavior before the beginning (basal) and 24h hours after the 3rd, 6th, 9th, 12th and 15th injections. Data are expressed as mean±S.E.M. (s). ANOVA with repeated measures revealed time, treatment and time x treatment interaction effects. *p<0.05 compared to VEH and RES 0.05 group (ANOVA and Bonferroni‘s test).
3.2- Effects of repeated administration of low doses of reserpine on plus-maze discriminative avoidance task
In the training session, we observed a significant effect of treatment in the distance traveled in the apparatus (Figure 3 A; ANOVA, F (3, 25) = 15.98, p = 0.000). The post hoc analysis (Duncan‘s test) revealed that RES 0.1 and 0.2 groups showed decreased motor activity when compared to groups VEH and RES 0.05 in the training session (24 h after administration of the 10th injection). In the test session, 48
0 20 40 60 80 100 0 3rd 6th 9th 12th 15th C atal ep sy du rat ion ( s)
55 h after administration of the 10th injection, there was no significant difference between groups in the distance traveled (Fig. 3 B; ANOVA, F (3, 25) = 0.51, p = 0.681).
Fig. 3: Effects of repeated administration of low doses of reserpine (RES - 0.05, 0.1 or 0.2 mg/Kg) or vehicle (VEH) on the plus-maze discriminative avoidance apparatus during training (A) and test (B) sessions performed 24 and 48 h after the 10th injection, respectively. * p <0.05 compared with vehicle and RES 0.05 group (ANOVA and Duncan‘s test)
In the training session (24h after the 10th injection), we found no significant differences in the percentage of time in the open arms (%TO) (Figure 4 A; ANOVA, F (3, 25) = 0.39, p = 0.761). However, in the test (48h after the 10th injection), a significant effect of treatment was found for %TO. The post hoc analysis (Duncan test) revealed that RES 0.2 group showed a significant increase in the percentage of time in open arms in the test session when compared with all other groups (Fig. 4 B, ANOVA, F (3, 25) = 8.99, p = 0.000).
56 Fig. 4: Effects of repeated administration of low doses of reserpine (RES - 0.05, 0.1 or 0.2 mg/Kg) or vehicle (VEH) on percent time in the open arms (%TO) of the plus- maze discriminative avoidance apparatus during training (A) and test (B) sessions, performed 24 and 48 h after the 10th injection, respectively. * p <0.05 compared with all other groups (ANOVA and Duncan‘s test)
Regarding the percent time in the aversive arm, we found no significant effects when the whole training session duration was considered for analysis (Figure 5 A, ANOVA, F (3, 25) = 1.99, p = 0. 142). When the same analysis was applied to the test session, we found a significant effect of treatment for percentage of time in the aversive arm. The post hoc analysis (Duncan test) showed that RES 0.1 group showed increased aversive arm exploration when compared with groups VEH and RES 0.05 (Figure 5 B, ANOVA, F (3, 25) = 3.25, p = 0. 039).
In the training session, significant effects of time (minutes) [F (9, 225)= 8.41; p=0.000] and the time X treatment interaction [F (9, 225)= 2.38; p=0.019] were found when the percentage of time in the aversive enclosed arm (%TAV) was evaluated across the session. No effect of the treatment (RES (0.05, 0.1 or 0.2 mg/Kg) or VEH) [F (3, 25)= 2.75; p=0.064] was found (see Fig 5 C).
57 In the test session, a significant effect of the time X treatment interaction [F (9, 225)= 2.04; p=0.029] was found when the percentage of time in the aversive enclosed arm (%TAV) was evaluated across the session. No effects of time (minutes) [F (9,225)= 1.38; p=0.246] or treatment (RES (0.05, 0.1 or 0.2 mg/Kg) or VEH) [F (3,25)= 0.96; p=0.429] were found (see Fig 5 D).
58 Fig. 5: Effects of repeated administration of low doses of reserpine (RES - 0.05, 0.1 or 0.2 mg/Kg) or vehicle (VEH) on the percent time spent in the aversive arm (%TAV) of the discriminative avoidance apparatus. Data are expressed as the mean±S.E.M. for the whole training (A) and test (B) sessions and minute by minute during training
59 (C) and test (D) sessions, performed 24 and 48 h after the 10th injection, respectively. ANOVA with repeated measures revealed time (minutes) effect, in training session, and time x treatment interaction effects, in training and test sessions (C and D)* p <0.05 compared with vehicle and RES 0.05 group (ANOVA and Duncan‘s test)
3.3- Effects of repeated administration of low doses of reserpine on orofacial movements
The analysis of the orofacial movements, held 24 h after the 14th injection, showed significant differences between groups for the number of vacuous chewing movements (Figure 6 A, F (3, 25) = 60.46, p = 0.000), the duration of facial twitching (Figure 6 B, F (3, 25) = 40.99, p = 0.000) and the number of tongue protrusions (Figure 6 C, F (3, 25) = 13.34, p = 0.000). The post hoc analysis (Duncan‘s test) showed that, in all orofacial movement measures, RES 0.1 and RES 0.2 groups