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Cineradiographic analysis of respiratory movements in a mouse model for early Parkinson's disease
Respiratory Physiology & Neurobiology 218 (2015) 40–45
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Cineradiographic analysis of respiratory movements in a mouse model for early Parkinson’s disease P.S. de Campos a , K. Hasegawa b , Y. Kumei c , J.L. Zeredo a,c,∗ a b c
Graduate Program in University of Brasilia, Health Sciences and Technologies, Brasilia, Brazil JAXA/Institute of Space and Astronautical Science, Sagamihara, Japan Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 5 June 2015 Accepted 3 July 2015 Available online 11 July 2015 Keywords: Respiration Diaphragm Radiography Disease models Behavior Animal
a b s t r a c t Parkinson’s disease (PD) is a progressive degenerative disorder of the central nervous system known to cause a typical pattern of motor symptoms. In its later stages, PD is known to cause respiratory alterations including shortening of operational volumes and reduced velocity of respiratory-muscle contraction. It has been proposed that such changes are secondary to changes in posture and osteoarticular degeneration, leading to an alteration in the spinal axis that in turn could affect breathing mechanics. In this study, we examined respiratory movements by using cineradiography on a murine (C57BL/6J) model of mild hemi-PD. Under surgical anesthesia, PD mice received an injection of 6-OHDA solution to the right striatum, and were compared to control mice, which received an injection of saline solution. Two weeks after surgery, all mice had their respiratory movements recorded by video X-ray without any restraint. Behavioral tests were performed to assess the severity of the 6-OHDA lesion. As a result, behavioral tests confirmed mild motor impairments in PD mice as compared to controls. Parameters of respiratory function showed mild alterations in the PD group, suggestive of a restrictive-type respiratory disorder. These results suggest that respiratory alterations in PD may emerge simultaneously to other motor symptoms, and not as a consequence of the latter. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a currently incurable, highly prevalent neurodegenerative disorder. PD affects mostly, but not exclusively, the elder population, with age being its the most important risk factor (Abdullah et al., 2014). The estimated prevalence is 0.2–0.3% in the general population and as much as 3% or more in persons over 65 years old (Errea et al., 1999; Khatter et al., 1996; Moghal et al., 1994). The prevalence decreases in the population above 90 years old, possibly because of increased mortality associated with the disease (Errea et al., 1999). PD is caused by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in well-known motor impairments that characterize the disease (Nicholson et al.,
Abbreviations: 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease; CPA, costophrenic angle. ∗ Corresponding author at: Graduate Program in Health Sciences and Technologies, University of Brasilia, Centro Metropolitano, Conjunto A, Lote 1, Brasília, DF 72220-900, Brazil. Tel.: +55 61 9975 9006. E-mail address: [email protected] (J.L. Zeredo). http://dx.doi.org/10.1016/j.resp.2015.07.002 1569-9048/© 2015 Elsevier B.V. All rights reserved.
2002). The diagnosis is essentially based on clinical criteria: resting tremor, muscle rigidity, and slowness of movements (bradykinesia), in addition to therapeutic response to levodopa (Tolosa et al., 2006). Diagnosing PD is straightforward when all clinical features are clearly present, which is not the case in the initial stages of the disease. In early PD, the first detected symptoms are peripheral tremors, especially of the hands, and at rest (Hoehn and Yahr, 1967). In mild to severe PD, in addition to worsening of the tremors, motor impairments gradually evolve to bradykinesia, akinesia, rigidity, and loss of postural reflexes. Further, as the disease progresses, many patients also develop other neurological impairments such as deficits of cognition (Koster et al., 2014). Respiratory symptoms are a common find in severe PD patients. However, it is less clear if such symptoms are present in the initial/mild stages of the disease. It is possible that respiratory symptoms are merely less evident because the other motor impairments reduce the demand on the respiratory system (Siderowf and Stern, 2008). Given that respiratory complications are the most common cause of death in PD patients (Nicholson et al., 2002), it is important to know the stage of the disease when these symptoms first emerge and if they are susceptible to early/preemptive treatments. Therefore, in this study we measured variables related to
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the respiratory function in the early stages of an animal model of PD. For this purpose a novel micro-focus X-ray movie system was employed. 2. Material and methods The methods used in this study followed the guidelines for animal welfare of the Tokyo Medical and Dental University and had institutional approval in advance of experimentation (Approval No. 0140089C). 2.1. Animals We used wildtype male C57BL/6J mice acquired from a commercial breeder (Nihon Clea, Tokyo, Japan). The animals were 8 weeks old (body weight: 23–25 g) at the beginning of experiments. The animals were housed individually in 20 cm × 20 cm × 30 cm acrylic cages lined with absorbing bedding material, with freely available food (standard chow) and water. A total of 22 mice were randomly divided in two groups, 6-OHDA (n = 14) and saline (n = 8). The animals were kept in a temperature controlled room (23 ± 1 ◦ C) and in a 12-h dark/light cycle (lights on at 8:00). 2.2. Surgical procedures Mice were anesthetized with a mixture of ketamine (90–120 mg/kg) and xylazine (10 mg/kg). Appropriate surgical level of anesthesia was verified by the lack of withdrawal reflexes from a pinch stimulus applied to the tail. The animals were then placed on a stereotaxic apparatus (Narishige, Tokyo, Japan) and received microinjections of either 6-OHDA or saline solutions into the striatum on the right side. The coordinates were AP: +0.5; L: −2.0 and DV: −3.0 mm (relative to bregma, sagittal suture, and dural surface) (Paxinos and Franklin, 2008). The solutions were prepared on the day of the surgery and kept in light-protected vials in an ice box until use. 6-OHDA solution consisted of 3 g 6-OHDA hydrochloride (Sigma Aldrich, Tokyo, Japan) dissolved in 0.9% NaCl with 0.02% ascorbic acid in sterile water. Saline solution consisted of 0.02% ascorbic acid and 0.9% NaCl in sterile water. For either solution the total injected volume was 2 L with the flow rate of 0.5 L/min. Injections were made through a 32 G needle attached to a 10 L Hamilton syringe on an injection pump. The injection needle was left in place for 2 min before and 2 min after the injection. The animals were monitored until recovered from anesthesia, then returned to their home cages. 2.3. Behavior tests Behavior tests were carried out 2 weeks after surgery. General motor function was assessed by the three tests described below. All behavior tests were scored by an examiner who was blind to the animals’ group assignment. Cylinder test: asymmetric paw preference in spontaneous exploratory behavior was examined by placing the mouse inside a glass beaker (8 cm diameter × 11 cm height) in front of vertical mirrors as to allow for a clear view of the mouse from all angles. The mice were filmed for 5 min while in the beaker. This test was performed only once, to avoid habituation, and just before lightsoff time, when the animals are most active. The normal animal behavior is to explore the new environment by rearing inside the beaker, touching the glass walls with its forepaws. The number of weight-bearing wall contacts made by the right and left forepaws was counted. Pole test: bradykinesia and motor coordination were assessed by placing the mice on top of a pole (50 cm in length, 1 cm in diameter) facing upwards. The normal animal behavior is to turn and climb
Fig. 1. Scheme of the cineradiography apparatus. A lateral view shows the test cage holding a mouse between the X-ray source and the image intensifier. The test cage can be gently moved by motors in the three dimensions, rotated, and tilted for optimal positioning of the mouse even as it moves in the cage. X-ray images were captured in real-time by a high-definition video camera.
down the pole. The mice were trained on this task before surgery, on two consecutive days. The test session was recorded on video and analyzed for the time spent to turn facing downwards, and to climb down the pole into the home-cage below. Each mouse completed the task three times and the average time was calculated. The cutoff time was 60 s. Nest-building test: motor activity and sensory-motor function were tested by placing squares of pressed cotton on the cage’s chow bin one hour before lights-off. Mice will use the cotton to build a nest overnight. The following morning, 12 h later, we collect and weight the amount of cotton material left unused in the bin, and calculated the amount that had been used out of an average initial weight of 2.67 g (±0.01 SD) of material. 2.4. Respiratory movements Respiratory movements in each mouse were recorded 2 weeks after surgery. Before the recordings, the animals were habituated to the cineradiographic apparatus (Micro X-movie, NIC, Fujisawa, Japan) for 5 min during two consecutive days. On the following two consecutive days, three 5-min recordings were made from each mouse. The animals were transferred one by one to a test cage inside the apparatus (Fig. 1). An X-ray beam was emitted vertically onto the unrestrained mouse by a micro-focus X-ray tube (Toshiba Electron Tubes and Devices Co. Ltd., Tokyo). Power on the X-ray tube was kept constant at 70 kV and 0.3 mA to obtain stable X-ray emission. The X-ray photons passing through mouse reached the 100 mm diameter entrance field of the high speed-response type beryllium image intensifier (Toshiba Electron Tubes and Devices Co., Ltd., E5889BP-P1K), which converted the X-ray photons into visible-light photons, creating an image that was captured by a digital video camera positioned underneath the image intensifier (Hasegawa et al., 2014). Video recordings were made at 29 fps and 1920 × 1080 pixels. We measured respiratory frequency, diaphragmatic excursion, and amplitude of the costophrenic angle (CPA) (details below). Three measurements were made from each recording, during quiet respiration, at the positions of maximum inspiration and the next
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3. Results
Fig. 2. Example of a cineradiographic video-still used for respiratory measurements. This picture shows the X-ray image of the mouse body in the horizontal plane. Dashed line outlines the thoracic cavity. Respiratory movements were measured by linear (height of the diaphragmatic dome), angular (variation of the costophrenic angle), and temporal (respiratory frequency) variables.
maximum expiration. Thus a total of 18 measurements were made for each mouse. Measurements were made by an examiner who was blind to whether the mice had received 6-OHDA or saline injection. Measurement of respiratory frequency was derived from the number of frames between maximum inspiration and the next maximum expiration. The height of the diaphragmatic dome was measured perpendicularly between the imaginary line connecting the right and left CPA and the highest point on the dome (Fig. 2). Diaphragmatic excursion was measured as the difference in the height of the diaphragmatic dome between maximum expiration and maximum inspiration. The CPA was measured between the edge of the chest wall and the highest point on the diaphragmatic dome on the right side of the body. The angular variation was obtained by subtracting the measurement on maximum inspiration by that on maximum expiration. The software Kinovea (http:// www.kinovea.org/) was used for frame-by-frame display and measurement of video images.
2.5. Statistical analysis Group differences between 6-OHDA and saline injected animals were compared by the Mann–Whitney U-test, where P ≤ 0.05 was considered statistically significant. Measurements are displayed as mean ± SEM.
Results of the behavioral tests were consistent with mild/initial Parkinsonism in the 6-OHDA mice as compared to saline (Fig. 3). In the cylinder test, 6-OHDA mice showed a preference to use the right forepaw than the left when rearing inside the cylinder (Fig. 3A). On average, 6-OHDA mice used the right forepaw 8.8 ± 3.7% more than the left one, while saline mice used the left forepaw 3.1 ± 3.9% more than the right one (P = 0.05). In the pole test, 6-OHDA mice took significantly longer to turn downwards before initiating the descent (Fig. 3B). The average time for turning down was 6.9 ± 1.9 s in the 6-OHDA group and 2.5 ± 0.2 s in the saline group (P = 0.02). On the other hand, the difference in the time spent to descent the 50-cm pole was not significant between groups. The average time for descent was 21.3 ± 2.3 s in the 6OHDA group and 16.3 ± 1.7 in the saline group (P = 0.58). In the nest-building test, the amount of material that the 6-OHDA mice used for nest-building was 0.6 ± 0.1 g, whereas that of the saline control mice was 1.0 ± 0.3 g (Fig. 3C). 6-OHDA did not significantly deteriorate the mouse nest-building activity (P = 0.68). During examination of X-ray images we found no signs of fibrosis or hyperinflation of the lungs in either 6-OHDA or saline group. There were no differences in the density of the lung image (as an indication of aspiration pneumonia) between groups. The respiratory movements showed no signs of mechanical or functional obstruction in either group. Measurements of respiratory movements showed significant differences between groups in one of the studied variables (Fig. 4). The respiratory frequency was similar between 6-OHDA and saline mice (Fig. 4A), with the averages of 2.91 ± 0.11 Hz and 2.80 ± 0.05 Hz, respectively (P = 0.39). In the 6-OHDA group, the range of respiratory frequencies was wider at 1.27 Hz as compared to the saline group at 0.48 Hz. Linear measurements of diaphragmatic excursion were similar between groups (Fig. 4B and C). The total displacement of the diaphragm during quiet respiration was in average 0.93 ± 0.02 mm in 6-OHDA mice and 0.98 ± 0.04 mm in saline mice (P = 0.29). The group ranges were similar at 0.36 mm and 0.33 mm, respectively. In the position of maximum expiration, the height of the diaphragmatic dome (as measured perpendicularly from a line between right and left costophrenic sinuses) was 4.06 ± 0.08 mm in 6-OHDA mice and 4.08 ± 0.13 mm in saline mice (P = 0.91). In the position of maximum inspiration, the height of the diaphragmatic dome was 3.15 ± 0.07 mm in 6-OHDA mice and 3.09 ± 0.12 mm in saline mice (P = 0.65). On the other hand, statistically significant differences were found in the measurements of the CPA. Although the angular difference of maximum inspiration and maximum expiration
Fig. 3. Behavioral tests. (A) Cylinder test results showed 6-OHDA mice with a significant bias toward using the side ipsilateral to the toxin injection site, as compared to saline mice. (B) In the pole test, 6-OHDA mice took significantly longer to turn downward and start the descent, but the total time to descend the pole was similar to saline mice. (C) Nest-building activity was similar between 6-OHDA and saline mice, without statistically significant intergroup difference. Values are means ± SEM. Asterisk denotes statistically significant difference (P ≤ 0.05) in the Mann–Whitney U-test.
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Fig. 4. Respiratory measurements. (A) 6-OHDA mice and saline mice showed similar respiratory frequencies during quiet respiration. The difference between groups was not statistically significant. (B) Total diaphragmatic excursion was measured as the difference in the height of the diaphragmatic dome between a maximum expiration and the next maximum inspiration, with similar results between groups. (C) Average height of the diaphragmatic dome was similar between groups during either expiration or inspiration. (D) Difference in the costophrenic angle from maximum inspiration to maximum expiration was similar between groups. (E) Costophrenic angle was significantly lower in 6-OHDA mice, as compared to saline, in both maximum expiration and maximum inspiration. Values are means ± SEM. Asterisk denotes statistically significant difference (P < 0.05) in the Mann–Whitney U-test.
was not significant between groups (Fig. 4D), 6-OHDA mice showed smaller CPA in both maximum expiration and maximum inspiration as compared to saline mice (Fig. 4E). The angular difference of maximum inspiration-maximum expiration was 7.41 ± 0.20◦ in 6-OHDA mice and 7.43 ± 0.14◦ in saline mice (P > 0.99). However, in maximum expiration the CPA was 46.29 ± 0.56◦ in 6-OHDA mice and 48 ± 0.85◦ in saline mice (P = 0.02), and in maximum inspiration the CPA was 53.47 ± 0.65◦ in 6-OHDA mice and 55.92 ± 0.91◦ in saline mice (P = 0.02). 4. Discussion In this study, we evaluated different components of the respiratory pattern during quiet respiration through cineradiography, in an animal model of PD mimicking the initial stages of the disease. We found that the CPA was lower in experimental animals as compared to controls. This alteration may be explained by the presence of chest wall and accessory respiratory-muscle rigidity, which are observed in respiratory disorders of the restrictive type. To the best of our knowledge, this is the first study to examine respiratory function in an animal model of PD. PD was simulated in mice by injecting the neurotoxin 6-OHDA unilaterally into the striatum. This model causes neurodegeneration of the nigrostrial dopaminergic system, which is known to induce the motor symptoms observed in PD patients (Damier et al., 1999; Gibb and Lees, 1991). Because the degenerative lesion
is unilateral in this animal model, the extension of the lesion can be evaluated by behavioral tests showing side bias in limb use/movement, as well as by general mobility and motor coordination (Francardo et al., 2011; Heuer et al., 2012; Iancu et al., 2005). In this study, a small but significant side bias was observed in the cylinder test in 6-OHDA injected mice. Previous reports have found that mice with a similar side bias magnitude had up to 65% of unilateral neuronal cell loss in the substantia nigra (Iancu et al., 2005). In patients, it is estimated that about 60% of nigrostriatal dopaminergic terminals bilaterally are already lost at the time of diagnosis (Jankovic and Sherer, 2014). Therefore, the experimental animals studied here may have been in a condition of motor impairment similar to that of patients in the initial, possibly even preclinical, stages of the disease. Dopamine mediates complex modulatory mechanisms for respiration within the CNS (Lalley, 2008). Previous studies indicate that the different dopamine receptor subtypes may have opposing effects on the respiratory drive: dopamine-D1 receptor activation increases respiratory neuron excitability (Lalley, 2004, 2009), and D2 and D4 effects are depressant (Pan et al., 2008), with a seemingly dominant D1 modulation (Lalley and Mifflin, 2012). This view is consistent with an overall reduction in respiratory efficiency in the condition of reduced dopamine availability. Mouse models of tauopathy, which encompasses neurodegenerative diseases such as Parkinson’s and Alzheimer’s, show important respiratory dysfunctions. Tau.P301L mice develop upper
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airway dysfunction at about 8 months of age and die prematurely at 12 months of age. Respiratory dysfunctions have been described as an airflow/chest rate of spirogram amplitude that is 1/3 that of wild type mice (Dutschmann et al., 2010), impaired ultrasonic vocalizations (Menuet et al., 2011), and prolonged chest electromyography (Menuet et al., 2012). Interestingly, Tau.P301L mice seem to compensate the tendency to upper airways closure during inspiration by increasing the movements of the chest (Dutschmann et al., 2010), a finding that is in line with the results from our experiments in 6-OHDA mice. Although respiration involves skeletal muscle contractions, and movements of bones and joints, respiratory problems are usually considered non-motor symptoms (Mehanna and Jankovic, 2010). Nevertheless, respiratory problems are a common find in patients with movements disorders such as PD (Nogués and Benarroch, 2008). Previous studies have reported respiratory disorders of both obstructive and restrictive type. Obstructive disorders are the most commonly reported type, with various possible mechanisms. Currently proposed pathophysiology includes deficits in the laryngeal-muscle function (Schwab et al., 1959; Smith et al., 1995), as well as disturbances in central feedback circuits in the basal nuclei for respiration and speech control (Hammer and Barlow, 2010; Ho et al., 2008). Muscle spasms (dystonia), can also cause respiratory obstruction when occurring on oral, pharyngeal, laryngeal, or cervical muscles (Jankovic and Tintner, 2001; Kato et al., 2007; Onoue et al., 2003). On the other hand, restrictive respiratory disorders are found less commonly (Mehanna and Jankovic, 2010; Sabaté et al., 1996b) and have been associated with advanced stages of PD. In this case, the pathophysiology has been attributed to respiratory muscle rigidity and bradykinesia, although there seems to be no clear relationship between pulmonary restrictive disorder and severity of motor symptoms in PD (Sabaté et al., 1996a). In addition, altered axial body posture in moderate to severe PD may also contribute to the restrictive respiratory disorder by limiting chest expansion during inspiration (Jankovic, 2010; Sabaté et al., 1996a). On the other hand, our findings suggest that a restrictive respiratory disorder may emerge simultaneously to bradykinesia, at the same stage of nigrostrial degeneration; therefore, we can speculate that these symptoms, respiratory and motor, may have a common pathophysiology of central origin, likely related to basal nuclei dysfunction. It should be noted that in this study both respiratory frequency and diaphragm mobility were unchanged in 6-OHDA mice. These results suggest that the restrictive pattern was limited to the chest wall without compromising the diaphragm. Previous studies have found that diaphragmatic function remains normal in PD patients, even when postural and respiratory abnormalities were present (Nugent et al., 1958; Petit and Delhez, 1961; Vercueil et al., 1999). Indeed, human studies indicate that respiratory alterations remain largely unnoticed in most PD patients until the disease is at an advanced stage (Hovestadt et al., 1989). These findings support the notion that respiratory alterations have complex and robust compensatory mechanisms that enable normal respiratory function in mild to moderate PD (Canning et al., 1997). On the other hand, considering the high mortality associated with respiratory symptoms (Nicholson et al., 2002), it seems advisable to begin a comprehensive respiratory rehabilitation program in PD patients as soon as a diagnosis is made.
5. Limitations of this study At present no single animal model can simulate all the complex features of PD (Bezard and Przedborski, 2011). Neurotoxin models such as the one used here are useful for the study of motor symptoms and development of symptomatic therapies; however,
they may not simulate PD-induced alterations unrelated to nigrostrial degeneration. In the present study, 6-OHDA was delivered into the striatum. This model induces a slow and progressive damage to the nigrostrial system retrogradely over a period of about 3 weeks (Da Conceic¸ão et al., 2010; Przedbroski et al., 1995; Sauer and Oertel, 1994). The lesion is progressive and less extensive than in other targets (nigra or medial forebrain bundle), which is considered to be more akin with PD (Tieu, 2011). Nevertheless, studies on the pathophysiology of premotor PD symptoms indicate that PD is likely to start not in the nigrostrial system, but in the dorsal motor nucleus of the glossopharyngeal and vagal nerves and anterior olfactory nucleus (Braak et al., 2003). Therefore, some of the premotor changes affecting respiratory movements may not have been detectable in the present experiments (Seccombe et al., 2011). In addition, C57BL/6J mice show the regular occurrence of spontaneous central apneas, caused by active laryngeal closure (Stettner et al., 2008). However, these apneas are known to be modulated by serotonin (Moore et al., 2012; Yamauchi et al., 2008), whose levels have been shown to remain similar to those of controls in mild 6OHDA models, at least in the striatum (Branchi et al., 2008), thus in principle both 6-OHDA and control animals would be equally prone to central apneas in this study. 6. Conclusions This study detected in PD model mice a decrease in thoracic amplitude, which indicates restrictive respiratory disorder. On the other hand, the respiratory rate and the amplitude of excursion of the diaphragm were similar to those in the control group, indicating normal diaphragmatic function at the initial stages of nigrostrial degeneration. These results suggest that respiratory alterations in PD may emerge simultaneously to other motor symptoms, and not as a consequence of the latter. Acknowledgments This work was financially supported by grants from JAXA (Japan) to Y. Kumei (FY2010-2011) and from JSPS (Japan) to Y. Kumei (24300190), J.L. Zeredo (23593021), and K. Hasegawa (23659885). P.S. de Campos presented a preliminary version of this work at the Neuroscience 2014 in Washington, DC with travel support from FINATEC (Brazil). References Abdullah, R., Basak, I., Patil, K.S., Alves, G., Larsen, J.P., Møller, S.G., 2014. Parkinson’s disease and age: the obvious but largely unexplored link. Exp. Gerontol., http:// dx.doi.org/10.1016/j.exger.2014.09.014 Bezard, E., Przedborski, S., 2011. A tale on animal models of Parkinson’s disease. Mov. Disord. 26, 993–1002, http://dx.doi.org/10.1002/mds.23696 Braak, H., Del Tredici, K., Rüb, U., de Vos, R.A.I., Jansen Steur, E.N.H., Braak, E., 2003. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging 24, 197–211. Branchi, I., D’Andrea, I., Armida, M., Cassano, T., Pèzzola, A., Potenza, R.L., Morgese, M.G., Popoli, P., Alleva, E., 2008. Nonmotor symptoms in Parkinson’s disease: investigating early-phase onset of behavioral dysfunction in the 6hydroxydopamine-lesioned rat model. J. Neurosci. Res. 86, 2050–2061, http:// dx.doi.org/10.1002/jnr.21642 Canning, C.G., Alison, J.A., Allen, N.E., Groeller, H., 1997. Parkinson’s disease: an investigation of exercise capacity, respiratory function, and gait. Arch. Phys. Med. Rehabil. 78, 199–207, http://dx.doi.org/10.1016/S0003-9993(97)90264-1 Da Conceic¸ão, F.S.L., Ngo-Abdalla, S., Houzel, J.-C., Rehen, S.K., 2010. Murine model for Parkinson’s disease: from 6-OH dopamine lesion to behavioral test. J. Vis. Exp., 9–11, http://dx.doi.org/10.3791/1376 Damier, P., Hirsch, E.C., Agid, Y., Graybiel, A.M., 1999. The substantia nigra of the human brain: II. Patterns of loss of dopamine-containing neurons in Parkinson’s disease. Brain 122, 1437–1448, http://dx.doi.org/10.1093/brain/122.8.1437 Dutschmann, M., Menuet, C., Stettner, G.M., Gestreau, C., Borghgraef, P., Devijver, H., Gielis, L., Hilaire, G., Van Leuven, F., 2010. Upper airway dysfunction of Tau-P301L mice correlates with tauopathy in midbrain and ponto-medullary brainstem nuclei. J. Neurosci. 30, 1810–1821, http://dx.doi.org/10.1523/JNEUROSCI.526109.2010
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Cineradiographic analysis of respiratory movements in a mouse model for early Parkinson’s disease P.S. de Campos a , K. Hasegawa b , Y. Kumei c , J.L. Zeredo a,c,∗ a b c
Graduate Program in University of Brasilia, Health Sciences and Technologies, Brasilia, Brazil JAXA/Institute of Space and Astronautical Science, Sagamihara, Japan Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 5 June 2015 Accepted 3 July 2015 Available online 11 July 2015 Keywords: Respiration Diaphragm Radiography Disease models Behavior Animal
a b s t r a c t Parkinson’s disease (PD) is a progressive degenerative disorder of the central nervous system known to cause a typical pattern of motor symptoms. In its later stages, PD is known to cause respiratory alterations including shortening of operational volumes and reduced velocity of respiratory-muscle contraction. It has been proposed that such changes are secondary to changes in posture and osteoarticular degeneration, leading to an alteration in the spinal axis that in turn could affect breathing mechanics. In this study, we examined respiratory movements by using cineradiography on a murine (C57BL/6J) model of mild hemi-PD. Under surgical anesthesia, PD mice received an injection of 6-OHDA solution to the right striatum, and were compared to control mice, which received an injection of saline solution. Two weeks after surgery, all mice had their respiratory movements recorded by video X-ray without any restraint. Behavioral tests were performed to assess the severity of the 6-OHDA lesion. As a result, behavioral tests confirmed mild motor impairments in PD mice as compared to controls. Parameters of respiratory function showed mild alterations in the PD group, suggestive of a restrictive-type respiratory disorder. These results suggest that respiratory alterations in PD may emerge simultaneously to other motor symptoms, and not as a consequence of the latter. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a currently incurable, highly prevalent neurodegenerative disorder. PD affects mostly, but not exclusively, the elder population, with age being its the most important risk factor (Abdullah et al., 2014). The estimated prevalence is 0.2–0.3% in the general population and as much as 3% or more in persons over 65 years old (Errea et al., 1999; Khatter et al., 1996; Moghal et al., 1994). The prevalence decreases in the population above 90 years old, possibly because of increased mortality associated with the disease (Errea et al., 1999). PD is caused by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in well-known motor impairments that characterize the disease (Nicholson et al.,
Abbreviations: 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease; CPA, costophrenic angle. ∗ Corresponding author at: Graduate Program in Health Sciences and Technologies, University of Brasilia, Centro Metropolitano, Conjunto A, Lote 1, Brasília, DF 72220-900, Brazil. Tel.: +55 61 9975 9006. E-mail address: [email protected] (J.L. Zeredo). http://dx.doi.org/10.1016/j.resp.2015.07.002 1569-9048/© 2015 Elsevier B.V. All rights reserved.
2002). The diagnosis is essentially based on clinical criteria: resting tremor, muscle rigidity, and slowness of movements (bradykinesia), in addition to therapeutic response to levodopa (Tolosa et al., 2006). Diagnosing PD is straightforward when all clinical features are clearly present, which is not the case in the initial stages of the disease. In early PD, the first detected symptoms are peripheral tremors, especially of the hands, and at rest (Hoehn and Yahr, 1967). In mild to severe PD, in addition to worsening of the tremors, motor impairments gradually evolve to bradykinesia, akinesia, rigidity, and loss of postural reflexes. Further, as the disease progresses, many patients also develop other neurological impairments such as deficits of cognition (Koster et al., 2014). Respiratory symptoms are a common find in severe PD patients. However, it is less clear if such symptoms are present in the initial/mild stages of the disease. It is possible that respiratory symptoms are merely less evident because the other motor impairments reduce the demand on the respiratory system (Siderowf and Stern, 2008). Given that respiratory complications are the most common cause of death in PD patients (Nicholson et al., 2002), it is important to know the stage of the disease when these symptoms first emerge and if they are susceptible to early/preemptive treatments. Therefore, in this study we measured variables related to
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the respiratory function in the early stages of an animal model of PD. For this purpose a novel micro-focus X-ray movie system was employed. 2. Material and methods The methods used in this study followed the guidelines for animal welfare of the Tokyo Medical and Dental University and had institutional approval in advance of experimentation (Approval No. 0140089C). 2.1. Animals We used wildtype male C57BL/6J mice acquired from a commercial breeder (Nihon Clea, Tokyo, Japan). The animals were 8 weeks old (body weight: 23–25 g) at the beginning of experiments. The animals were housed individually in 20 cm × 20 cm × 30 cm acrylic cages lined with absorbing bedding material, with freely available food (standard chow) and water. A total of 22 mice were randomly divided in two groups, 6-OHDA (n = 14) and saline (n = 8). The animals were kept in a temperature controlled room (23 ± 1 ◦ C) and in a 12-h dark/light cycle (lights on at 8:00). 2.2. Surgical procedures Mice were anesthetized with a mixture of ketamine (90–120 mg/kg) and xylazine (10 mg/kg). Appropriate surgical level of anesthesia was verified by the lack of withdrawal reflexes from a pinch stimulus applied to the tail. The animals were then placed on a stereotaxic apparatus (Narishige, Tokyo, Japan) and received microinjections of either 6-OHDA or saline solutions into the striatum on the right side. The coordinates were AP: +0.5; L: −2.0 and DV: −3.0 mm (relative to bregma, sagittal suture, and dural surface) (Paxinos and Franklin, 2008). The solutions were prepared on the day of the surgery and kept in light-protected vials in an ice box until use. 6-OHDA solution consisted of 3 g 6-OHDA hydrochloride (Sigma Aldrich, Tokyo, Japan) dissolved in 0.9% NaCl with 0.02% ascorbic acid in sterile water. Saline solution consisted of 0.02% ascorbic acid and 0.9% NaCl in sterile water. For either solution the total injected volume was 2 L with the flow rate of 0.5 L/min. Injections were made through a 32 G needle attached to a 10 L Hamilton syringe on an injection pump. The injection needle was left in place for 2 min before and 2 min after the injection. The animals were monitored until recovered from anesthesia, then returned to their home cages. 2.3. Behavior tests Behavior tests were carried out 2 weeks after surgery. General motor function was assessed by the three tests described below. All behavior tests were scored by an examiner who was blind to the animals’ group assignment. Cylinder test: asymmetric paw preference in spontaneous exploratory behavior was examined by placing the mouse inside a glass beaker (8 cm diameter × 11 cm height) in front of vertical mirrors as to allow for a clear view of the mouse from all angles. The mice were filmed for 5 min while in the beaker. This test was performed only once, to avoid habituation, and just before lightsoff time, when the animals are most active. The normal animal behavior is to explore the new environment by rearing inside the beaker, touching the glass walls with its forepaws. The number of weight-bearing wall contacts made by the right and left forepaws was counted. Pole test: bradykinesia and motor coordination were assessed by placing the mice on top of a pole (50 cm in length, 1 cm in diameter) facing upwards. The normal animal behavior is to turn and climb
Fig. 1. Scheme of the cineradiography apparatus. A lateral view shows the test cage holding a mouse between the X-ray source and the image intensifier. The test cage can be gently moved by motors in the three dimensions, rotated, and tilted for optimal positioning of the mouse even as it moves in the cage. X-ray images were captured in real-time by a high-definition video camera.
down the pole. The mice were trained on this task before surgery, on two consecutive days. The test session was recorded on video and analyzed for the time spent to turn facing downwards, and to climb down the pole into the home-cage below. Each mouse completed the task three times and the average time was calculated. The cutoff time was 60 s. Nest-building test: motor activity and sensory-motor function were tested by placing squares of pressed cotton on the cage’s chow bin one hour before lights-off. Mice will use the cotton to build a nest overnight. The following morning, 12 h later, we collect and weight the amount of cotton material left unused in the bin, and calculated the amount that had been used out of an average initial weight of 2.67 g (±0.01 SD) of material. 2.4. Respiratory movements Respiratory movements in each mouse were recorded 2 weeks after surgery. Before the recordings, the animals were habituated to the cineradiographic apparatus (Micro X-movie, NIC, Fujisawa, Japan) for 5 min during two consecutive days. On the following two consecutive days, three 5-min recordings were made from each mouse. The animals were transferred one by one to a test cage inside the apparatus (Fig. 1). An X-ray beam was emitted vertically onto the unrestrained mouse by a micro-focus X-ray tube (Toshiba Electron Tubes and Devices Co. Ltd., Tokyo). Power on the X-ray tube was kept constant at 70 kV and 0.3 mA to obtain stable X-ray emission. The X-ray photons passing through mouse reached the 100 mm diameter entrance field of the high speed-response type beryllium image intensifier (Toshiba Electron Tubes and Devices Co., Ltd., E5889BP-P1K), which converted the X-ray photons into visible-light photons, creating an image that was captured by a digital video camera positioned underneath the image intensifier (Hasegawa et al., 2014). Video recordings were made at 29 fps and 1920 × 1080 pixels. We measured respiratory frequency, diaphragmatic excursion, and amplitude of the costophrenic angle (CPA) (details below). Three measurements were made from each recording, during quiet respiration, at the positions of maximum inspiration and the next
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3. Results
Fig. 2. Example of a cineradiographic video-still used for respiratory measurements. This picture shows the X-ray image of the mouse body in the horizontal plane. Dashed line outlines the thoracic cavity. Respiratory movements were measured by linear (height of the diaphragmatic dome), angular (variation of the costophrenic angle), and temporal (respiratory frequency) variables.
maximum expiration. Thus a total of 18 measurements were made for each mouse. Measurements were made by an examiner who was blind to whether the mice had received 6-OHDA or saline injection. Measurement of respiratory frequency was derived from the number of frames between maximum inspiration and the next maximum expiration. The height of the diaphragmatic dome was measured perpendicularly between the imaginary line connecting the right and left CPA and the highest point on the dome (Fig. 2). Diaphragmatic excursion was measured as the difference in the height of the diaphragmatic dome between maximum expiration and maximum inspiration. The CPA was measured between the edge of the chest wall and the highest point on the diaphragmatic dome on the right side of the body. The angular variation was obtained by subtracting the measurement on maximum inspiration by that on maximum expiration. The software Kinovea (http:// www.kinovea.org/) was used for frame-by-frame display and measurement of video images.
2.5. Statistical analysis Group differences between 6-OHDA and saline injected animals were compared by the Mann–Whitney U-test, where P ≤ 0.05 was considered statistically significant. Measurements are displayed as mean ± SEM.
Results of the behavioral tests were consistent with mild/initial Parkinsonism in the 6-OHDA mice as compared to saline (Fig. 3). In the cylinder test, 6-OHDA mice showed a preference to use the right forepaw than the left when rearing inside the cylinder (Fig. 3A). On average, 6-OHDA mice used the right forepaw 8.8 ± 3.7% more than the left one, while saline mice used the left forepaw 3.1 ± 3.9% more than the right one (P = 0.05). In the pole test, 6-OHDA mice took significantly longer to turn downwards before initiating the descent (Fig. 3B). The average time for turning down was 6.9 ± 1.9 s in the 6-OHDA group and 2.5 ± 0.2 s in the saline group (P = 0.02). On the other hand, the difference in the time spent to descent the 50-cm pole was not significant between groups. The average time for descent was 21.3 ± 2.3 s in the 6OHDA group and 16.3 ± 1.7 in the saline group (P = 0.58). In the nest-building test, the amount of material that the 6-OHDA mice used for nest-building was 0.6 ± 0.1 g, whereas that of the saline control mice was 1.0 ± 0.3 g (Fig. 3C). 6-OHDA did not significantly deteriorate the mouse nest-building activity (P = 0.68). During examination of X-ray images we found no signs of fibrosis or hyperinflation of the lungs in either 6-OHDA or saline group. There were no differences in the density of the lung image (as an indication of aspiration pneumonia) between groups. The respiratory movements showed no signs of mechanical or functional obstruction in either group. Measurements of respiratory movements showed significant differences between groups in one of the studied variables (Fig. 4). The respiratory frequency was similar between 6-OHDA and saline mice (Fig. 4A), with the averages of 2.91 ± 0.11 Hz and 2.80 ± 0.05 Hz, respectively (P = 0.39). In the 6-OHDA group, the range of respiratory frequencies was wider at 1.27 Hz as compared to the saline group at 0.48 Hz. Linear measurements of diaphragmatic excursion were similar between groups (Fig. 4B and C). The total displacement of the diaphragm during quiet respiration was in average 0.93 ± 0.02 mm in 6-OHDA mice and 0.98 ± 0.04 mm in saline mice (P = 0.29). The group ranges were similar at 0.36 mm and 0.33 mm, respectively. In the position of maximum expiration, the height of the diaphragmatic dome (as measured perpendicularly from a line between right and left costophrenic sinuses) was 4.06 ± 0.08 mm in 6-OHDA mice and 4.08 ± 0.13 mm in saline mice (P = 0.91). In the position of maximum inspiration, the height of the diaphragmatic dome was 3.15 ± 0.07 mm in 6-OHDA mice and 3.09 ± 0.12 mm in saline mice (P = 0.65). On the other hand, statistically significant differences were found in the measurements of the CPA. Although the angular difference of maximum inspiration and maximum expiration
Fig. 3. Behavioral tests. (A) Cylinder test results showed 6-OHDA mice with a significant bias toward using the side ipsilateral to the toxin injection site, as compared to saline mice. (B) In the pole test, 6-OHDA mice took significantly longer to turn downward and start the descent, but the total time to descend the pole was similar to saline mice. (C) Nest-building activity was similar between 6-OHDA and saline mice, without statistically significant intergroup difference. Values are means ± SEM. Asterisk denotes statistically significant difference (P ≤ 0.05) in the Mann–Whitney U-test.
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Fig. 4. Respiratory measurements. (A) 6-OHDA mice and saline mice showed similar respiratory frequencies during quiet respiration. The difference between groups was not statistically significant. (B) Total diaphragmatic excursion was measured as the difference in the height of the diaphragmatic dome between a maximum expiration and the next maximum inspiration, with similar results between groups. (C) Average height of the diaphragmatic dome was similar between groups during either expiration or inspiration. (D) Difference in the costophrenic angle from maximum inspiration to maximum expiration was similar between groups. (E) Costophrenic angle was significantly lower in 6-OHDA mice, as compared to saline, in both maximum expiration and maximum inspiration. Values are means ± SEM. Asterisk denotes statistically significant difference (P < 0.05) in the Mann–Whitney U-test.
was not significant between groups (Fig. 4D), 6-OHDA mice showed smaller CPA in both maximum expiration and maximum inspiration as compared to saline mice (Fig. 4E). The angular difference of maximum inspiration-maximum expiration was 7.41 ± 0.20◦ in 6-OHDA mice and 7.43 ± 0.14◦ in saline mice (P > 0.99). However, in maximum expiration the CPA was 46.29 ± 0.56◦ in 6-OHDA mice and 48 ± 0.85◦ in saline mice (P = 0.02), and in maximum inspiration the CPA was 53.47 ± 0.65◦ in 6-OHDA mice and 55.92 ± 0.91◦ in saline mice (P = 0.02). 4. Discussion In this study, we evaluated different components of the respiratory pattern during quiet respiration through cineradiography, in an animal model of PD mimicking the initial stages of the disease. We found that the CPA was lower in experimental animals as compared to controls. This alteration may be explained by the presence of chest wall and accessory respiratory-muscle rigidity, which are observed in respiratory disorders of the restrictive type. To the best of our knowledge, this is the first study to examine respiratory function in an animal model of PD. PD was simulated in mice by injecting the neurotoxin 6-OHDA unilaterally into the striatum. This model causes neurodegeneration of the nigrostrial dopaminergic system, which is known to induce the motor symptoms observed in PD patients (Damier et al., 1999; Gibb and Lees, 1991). Because the degenerative lesion
is unilateral in this animal model, the extension of the lesion can be evaluated by behavioral tests showing side bias in limb use/movement, as well as by general mobility and motor coordination (Francardo et al., 2011; Heuer et al., 2012; Iancu et al., 2005). In this study, a small but significant side bias was observed in the cylinder test in 6-OHDA injected mice. Previous reports have found that mice with a similar side bias magnitude had up to 65% of unilateral neuronal cell loss in the substantia nigra (Iancu et al., 2005). In patients, it is estimated that about 60% of nigrostriatal dopaminergic terminals bilaterally are already lost at the time of diagnosis (Jankovic and Sherer, 2014). Therefore, the experimental animals studied here may have been in a condition of motor impairment similar to that of patients in the initial, possibly even preclinical, stages of the disease. Dopamine mediates complex modulatory mechanisms for respiration within the CNS (Lalley, 2008). Previous studies indicate that the different dopamine receptor subtypes may have opposing effects on the respiratory drive: dopamine-D1 receptor activation increases respiratory neuron excitability (Lalley, 2004, 2009), and D2 and D4 effects are depressant (Pan et al., 2008), with a seemingly dominant D1 modulation (Lalley and Mifflin, 2012). This view is consistent with an overall reduction in respiratory efficiency in the condition of reduced dopamine availability. Mouse models of tauopathy, which encompasses neurodegenerative diseases such as Parkinson’s and Alzheimer’s, show important respiratory dysfunctions. Tau.P301L mice develop upper
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airway dysfunction at about 8 months of age and die prematurely at 12 months of age. Respiratory dysfunctions have been described as an airflow/chest rate of spirogram amplitude that is 1/3 that of wild type mice (Dutschmann et al., 2010), impaired ultrasonic vocalizations (Menuet et al., 2011), and prolonged chest electromyography (Menuet et al., 2012). Interestingly, Tau.P301L mice seem to compensate the tendency to upper airways closure during inspiration by increasing the movements of the chest (Dutschmann et al., 2010), a finding that is in line with the results from our experiments in 6-OHDA mice. Although respiration involves skeletal muscle contractions, and movements of bones and joints, respiratory problems are usually considered non-motor symptoms (Mehanna and Jankovic, 2010). Nevertheless, respiratory problems are a common find in patients with movements disorders such as PD (Nogués and Benarroch, 2008). Previous studies have reported respiratory disorders of both obstructive and restrictive type. Obstructive disorders are the most commonly reported type, with various possible mechanisms. Currently proposed pathophysiology includes deficits in the laryngeal-muscle function (Schwab et al., 1959; Smith et al., 1995), as well as disturbances in central feedback circuits in the basal nuclei for respiration and speech control (Hammer and Barlow, 2010; Ho et al., 2008). Muscle spasms (dystonia), can also cause respiratory obstruction when occurring on oral, pharyngeal, laryngeal, or cervical muscles (Jankovic and Tintner, 2001; Kato et al., 2007; Onoue et al., 2003). On the other hand, restrictive respiratory disorders are found less commonly (Mehanna and Jankovic, 2010; Sabaté et al., 1996b) and have been associated with advanced stages of PD. In this case, the pathophysiology has been attributed to respiratory muscle rigidity and bradykinesia, although there seems to be no clear relationship between pulmonary restrictive disorder and severity of motor symptoms in PD (Sabaté et al., 1996a). In addition, altered axial body posture in moderate to severe PD may also contribute to the restrictive respiratory disorder by limiting chest expansion during inspiration (Jankovic, 2010; Sabaté et al., 1996a). On the other hand, our findings suggest that a restrictive respiratory disorder may emerge simultaneously to bradykinesia, at the same stage of nigrostrial degeneration; therefore, we can speculate that these symptoms, respiratory and motor, may have a common pathophysiology of central origin, likely related to basal nuclei dysfunction. It should be noted that in this study both respiratory frequency and diaphragm mobility were unchanged in 6-OHDA mice. These results suggest that the restrictive pattern was limited to the chest wall without compromising the diaphragm. Previous studies have found that diaphragmatic function remains normal in PD patients, even when postural and respiratory abnormalities were present (Nugent et al., 1958; Petit and Delhez, 1961; Vercueil et al., 1999). Indeed, human studies indicate that respiratory alterations remain largely unnoticed in most PD patients until the disease is at an advanced stage (Hovestadt et al., 1989). These findings support the notion that respiratory alterations have complex and robust compensatory mechanisms that enable normal respiratory function in mild to moderate PD (Canning et al., 1997). On the other hand, considering the high mortality associated with respiratory symptoms (Nicholson et al., 2002), it seems advisable to begin a comprehensive respiratory rehabilitation program in PD patients as soon as a diagnosis is made.
5. Limitations of this study At present no single animal model can simulate all the complex features of PD (Bezard and Przedborski, 2011). Neurotoxin models such as the one used here are useful for the study of motor symptoms and development of symptomatic therapies; however,
they may not simulate PD-induced alterations unrelated to nigrostrial degeneration. In the present study, 6-OHDA was delivered into the striatum. This model induces a slow and progressive damage to the nigrostrial system retrogradely over a period of about 3 weeks (Da Conceic¸ão et al., 2010; Przedbroski et al., 1995; Sauer and Oertel, 1994). The lesion is progressive and less extensive than in other targets (nigra or medial forebrain bundle), which is considered to be more akin with PD (Tieu, 2011). Nevertheless, studies on the pathophysiology of premotor PD symptoms indicate that PD is likely to start not in the nigrostrial system, but in the dorsal motor nucleus of the glossopharyngeal and vagal nerves and anterior olfactory nucleus (Braak et al., 2003). Therefore, some of the premotor changes affecting respiratory movements may not have been detectable in the present experiments (Seccombe et al., 2011). In addition, C57BL/6J mice show the regular occurrence of spontaneous central apneas, caused by active laryngeal closure (Stettner et al., 2008). However, these apneas are known to be modulated by serotonin (Moore et al., 2012; Yamauchi et al., 2008), whose levels have been shown to remain similar to those of controls in mild 6OHDA models, at least in the striatum (Branchi et al., 2008), thus in principle both 6-OHDA and control animals would be equally prone to central apneas in this study. 6. Conclusions This study detected in PD model mice a decrease in thoracic amplitude, which indicates restrictive respiratory disorder. On the other hand, the respiratory rate and the amplitude of excursion of the diaphragm were similar to those in the control group, indicating normal diaphragmatic function at the initial stages of nigrostrial degeneration. These results suggest that respiratory alterations in PD may emerge simultaneously to other motor symptoms, and not as a consequence of the latter. Acknowledgments This work was financially supported by grants from JAXA (Japan) to Y. Kumei (FY2010-2011) and from JSPS (Japan) to Y. Kumei (24300190), J.L. Zeredo (23593021), and K. Hasegawa (23659885). P.S. de Campos presented a preliminary version of this work at the Neuroscience 2014 in Washington, DC with travel support from FINATEC (Brazil). References Abdullah, R., Basak, I., Patil, K.S., Alves, G., Larsen, J.P., Møller, S.G., 2014. Parkinson’s disease and age: the obvious but largely unexplored link. Exp. Gerontol., http:// dx.doi.org/10.1016/j.exger.2014.09.014 Bezard, E., Przedborski, S., 2011. A tale on animal models of Parkinson’s disease. Mov. Disord. 26, 993–1002, http://dx.doi.org/10.1002/mds.23696 Braak, H., Del Tredici, K., Rüb, U., de Vos, R.A.I., Jansen Steur, E.N.H., Braak, E., 2003. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging 24, 197–211. Branchi, I., D’Andrea, I., Armida, M., Cassano, T., Pèzzola, A., Potenza, R.L., Morgese, M.G., Popoli, P., Alleva, E., 2008. Nonmotor symptoms in Parkinson’s disease: investigating early-phase onset of behavioral dysfunction in the 6hydroxydopamine-lesioned rat model. J. Neurosci. Res. 86, 2050–2061, http:// dx.doi.org/10.1002/jnr.21642 Canning, C.G., Alison, J.A., Allen, N.E., Groeller, H., 1997. Parkinson’s disease: an investigation of exercise capacity, respiratory function, and gait. Arch. Phys. Med. Rehabil. 78, 199–207, http://dx.doi.org/10.1016/S0003-9993(97)90264-1 Da Conceic¸ão, F.S.L., Ngo-Abdalla, S., Houzel, J.-C., Rehen, S.K., 2010. Murine model for Parkinson’s disease: from 6-OH dopamine lesion to behavioral test. J. Vis. Exp., 9–11, http://dx.doi.org/10.3791/1376 Damier, P., Hirsch, E.C., Agid, Y., Graybiel, A.M., 1999. The substantia nigra of the human brain: II. Patterns of loss of dopamine-containing neurons in Parkinson’s disease. Brain 122, 1437–1448, http://dx.doi.org/10.1093/brain/122.8.1437 Dutschmann, M., Menuet, C., Stettner, G.M., Gestreau, C., Borghgraef, P., Devijver, H., Gielis, L., Hilaire, G., Van Leuven, F., 2010. Upper airway dysfunction of Tau-P301L mice correlates with tauopathy in midbrain and ponto-medullary brainstem nuclei. J. Neurosci. 30, 1810–1821, http://dx.doi.org/10.1523/JNEUROSCI.526109.2010
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