Forced nonuse in unilateral parkinsonian rats exacerbates injury

Abstract

Diagnosis of Parkinson's disease (PD) is based on the presentation of clinical symptoms such as bradykinesia, resting tremor, and rigidity. However, one feature of PD that often begins years before diagnosis is decreased physical activity. We hypothesized that this depressed activity is not only a symptom of the early dopaminergic loss but also a catalyst in the degenerative process. Two experiments were performed to test this hypothesis. First, rats were exposed to a mild dose of 6-hydroxydopamine unilaterally into the nigrostriatal dopamine (DA) projections, which would normally result in an approximately 20% DA loss and no detectable behavioral asymmetries. A subset of these lesioned animals then had a cast applied for 7 d to the contralateral forelimb. After the cast was removed, these animals displayed long-term behavioral asymmetry and exacerbation of neurochemical loss (approximately 60% depletion). Second, a group of animals received a high dose of 6-hydroxydopamine that normally would yield a severe loss of nigrostriatal terminals (approximately 90% loss) and chronic sensorimotor deficits. During the first 7 d after neurotoxin exposure, a subset of these animals were forced to rely on the contralateral forelimb, a procedure we have previously reported to protect DA terminals and behavioral function. Some of these rats then had the use of their "recovered" forelimb restricted during the second or third week after lesioning. This precipitated a severe and chronic loss of DA terminals and functional deficits. These results suggest decreased physical activity not only is a symptom of PD but also may act to potentiate the underlying degeneration.

Fig. 1.

Behavioral asymmetries after forced nonuse in animals with mild lesions. A, Animals given mild unilateral lesions did not display significant limb use asymmetry. When animals were forced to not use the impaired forelimb for the first 7 d after lesioning, they demonstrated limb use asymmetry that persisted across testing days. B, Animals given mild unilateral lesions did not display significant forelimb akinesia. When animals were forced to not use the impaired forelimb for the first 7 d after lesioning, they demonstrated forelimb akinesia that persisted across testing days. C, Animals given mild unilateral lesions did suffer placing deficits. When animals were forced to not use the impaired forelimb for the first 7 d after lesioning, they demonstrated significant placing deficits that persisted across testing days (*p < 0.01 compared with sham controls; ⁺p < 0.01 compared with lesion only).

Fig. 2.

Effect of forced nonuse of the impaired forelimb after mild 6-OHDA lesion. A, A 5 μg infusion of 6-OHDA resulted in only a mild loss of DA and HVA in striatal tissue when values were compared with the intact hemisphere. In contrast, forced nonuse of the impaired forelimb for the first 7 d after lesioning resulted in significantly greater loss of DA and its metabolites when compared with both sham animals and animals lesioned but not casted; *p < 0.05 compared with sham; ⁺p < 0.01 compared with lesion/no cast. B, Immunoreactivity of DAT, VMAT2, and TH was not reduced after mild lesioning (calculated as percentage remaining in lesion hemisphere). In contrast, forced nonuse of the impaired forelimb for the first 7 d after lesioning resulted in significant declines in DAT, VMAT2, and TH immunoreactivity; *p < 0.01 compared with sham;⁺p < 0.02 compared with lesion/no cast.C, Representative blots of VMAT2, DAT, and TH for sham, mild lesion, and mild lesion and nonuse groups. Ctrl, Control; Les, lesion.

Fig. 3.

Behavioral asymmetries after forced nonuse in animals with severe DA lesions. A, Animals forced to rely on the impaired forelimb for the first 7 d after severe unilateral lesioning did not display characteristic limb use asymmetry. In contrast, when animals were later forced to not use the impaired forelimb after recovery induced by forced use, asymmetrical limb use characteristic of the high dose of 6-OHDA received at the time of surgery was observed (*p < 0.01). B, Animals forced to rely on the impaired forelimb for the first 7 d after severe unilateral lesioning did not display characteristic forelimb akinesia. In contrast, when animals were later forced to not use the impaired forelimb after recovery induced by forced use, marked forelimb akinesia characteristic of the amount of neurotoxin sustained during surgery was observed (*p < 0.01).C, Animals forced to rely on the impaired forelimb for the first 7 d after severe unilateral lesioning did not display characteristic placing deficits. In contrast, when animals were later forced to not use the impaired forelimb after forced use-induced recovery, occurrence or reinstatement of placing inability was observed (*p < 0.01).

Fig. 4.

Apomorphine-induced rotation after forced nonuse in animals with recovery of function. A, Unilateral 10 μg infusion of 6-OHDA resulted in significant contralateral rotation after apomorphine administration 18 d after lesioning. Forced use of the impaired forelimb for the first 7 d after lesioning resulted in an absence of apomorphine-induced rotation. When animals were forced to not use the limb on days 7–14 after a 7 d period of forced use, they displayed significant apomorphine rotation.B, Animals forced to rely on the impaired forelimb for the first 7 d after lesioning and later had the impaired forelimb casted on days 21–28 did not display significant apomorphine-induced rotation 18 d after lesioning. In contrast, when tested on day 35 after lesioning (after the period of nonuse), these animals displayed significant contralateral rotation (*p < 0.05 compared with shams; ⁺p < 0.01 day 18).

Fig. 5.

Effect of forced nonuse of the impaired forelimb after severe 6-OHDA lesion. A, A 10 μg infusion of 6-OHDA resulted in severe lesioning as reflected by significant decrease in the percentage of DA, DOPAC, and HVA remaining. Forced use of the impaired forelimb for the first 7 d after lesioning resulted in amelioration of these losses, but subsequent nonuse of the impaired forelimb on days 7–14 or 21–28 resulted in a reinstatement of lesion induced losses; *p < 0.01 compared with sham.B, Immunoreactivity of DAT, VMAT2, and TH was significantly reduced after a 10 μg unilateral lesion, but the reactivity of these proteins was significantly increased after forced use of the impaired forelimb for the first 7 d after lesioning. A subsequent period of nonuse after forced use resulted in decreased immunoreactivity of DAT, VMAT2, and TH (*p < 0.01 sham;⁺p < 0.01 lesion only).C, Representative blots of VMAT2, DAT, and TH for animals subjected to a severe lesion only, severe lesion plus forced use on postoperative days 1–7, and severe lesion plus forced use on days 1–7 followed by nonuse on days 21–28. Ctrl, Control; Les, lesion.