Animal preparation

Adult male Long-Evans rats were used. Animals were food deprived for 12 hours prior to surgery and were maintained at 37° for all surgical procedures.

90 min 2 Vessel Occlusion (2VO)

Animals were anesthetized with chloral hydrate (49 mg/kg IP). The left middle cerebral artery (MCA) was exposed by drilling through the squamous temporal bone midway between the right eye and ear. After incision of the dura mater, a wire occluder (.005”) was introduced under the MCA, lifted gently, and turned 90°. Interruption of blood flow was confirmed using a Laser Doppler Flowmeter (LDF). The ipsilateral common carotid artery (CCA), which had been isolated via a neck incision, was occluded using atraumatic aneurism clips. At the end of the 90 min ischemic period, blood flow was reestablished and confirmed with LDF. Animals were casted for either:

3 days and sacrificed at 3 days 14 days and sacrificed at 28 days

30 min 3 Vessel Occlusion (3VO)

Anesthesia and surgeries were performed as for 90 min 2VO, but the left MCA and both CCAs were occluded. Blood flow was reestablished at the end of the 30 min ischemic period and confirmed by LDF. Animals were casted for 10 days and sacrificed at 32 days.

6-OHDA infusion

Animals were anesthetized with Equithesin (.035 ml/kg). 10 µg 6-OHDA (2.5 µg/ml in 0.1% ascorbic acid) was infused throgh a cannula into medial forebrain bundle using the coordinates of Paxinos and Watson: A/P -3.3; L/M +/- 1.7; D/V -9. Animals were casted for 7 days and sacrificed at 60 days.

Casting procedure

Prior to recovery from anesthesia, animals were either fitted with one sleeved plaster casts, as shown in Figure 1, or left uncasted. The upper torso was wrapped in soft felt, and the ipsilateral forelimb was wrapped in felt and positioned in a naturally retracted position against the animal's sternum. Plaster of paris strips were then wrapped around the immobilized limb and upper torso.

Fig. 1: Rats were fitted with one-sleeved plaster of Paris casts.

Behavioral Tests

Forelimb placing

Each animal was held by the torso with forelimbs hanging freely. Contralateral and ipsilateral placing was induced by gently brushing the respective vibrissae on the edge of a tabletop 10 times. Contralateral results are shown (ipsilateral placing was 100% for all groups) as percent successful placing (number correct x 10).

Footfault

Each animal was placed on an elevated grid for 2 min. Each time a paw slipped through the grid, as the animal explored, a footfault was scored. Total number of steps was also counted. Footfault index was computed as (ipsilateral faults - contralateral faults)/(total steps). A score of 0 = no asymmetry.

Limb use asymmetry

Each animal was placed in a clear glass cylinder for 5 min and videotaped. Videotapes were analyzed in slow motion, and scored for placement of forepaws against the side of the cylinder as the animal reared. Ipsilateral, contralateral and simultaneous placements were tallied. Limb use index was computed as (ipsilateral - contralateral)/total. A score of 0 = no asymmetry.

Apomorphine rotation

Apomorphine (.25 mg/kg in 0.1% ascorbic acid) was injected subcutaneously. Animals were returned to their home cages, and contralateral turns were tallied for 90 minutes.

Histology

Animals in the 90 min 2VO experiment were sacrificed at either 3 days or 28 days following surgery by intracardiac perfusion with 0.9% saline. The brains were removed, sliced into seven 2 mm slices, and stained with 2% triphenly tetrazolium chloride (TTC).

Animals in the 6-OHDA experiment were sacrificed at 60 days by sodium pentobarbital overdose. The brains were removed, and striatum and substantia nigra were dissected and rapidly frozen for subsequent quantification of tyrosine hydroxylase.

Forced motor therapy resulted in an increase in infarct volume following 90 min 2VO, as can be seen in Figure 2. Infarct volumes were larger in rats casted for 3 days and sacrificed at 3 days (A) and in rats casted for 14 days and sacrificed at 28 days (B).

Fig. 2: Infarct volume 3 days and 28 days following 90 min 2VO.

Forced motor therapy resulted in exaggerated behavioral deficits following 30 min 3VO, as can be seen in Figure 3. Rats that had been casted were more impaired in forelimb placing (top) and footfault (bottom) than uncasted rats or shams.

Fig. 3: Forelimb placing and footfault scores following 30 min 3VO.

Forced motor therapy resulted in fewer apomorphine induced contralateral rotations in 6-OHDA treated rats, as can be seen in Figure 4.

Fig. 4: Apomorphine rotation in 6-OHDA treated rats.

Forced motor therapy resulted in decreases in spontaneous limb use asymmetries in 6-OHDA treated rats, as can be seen in Figure 5. Rats that had been casted were similar to sham controls.

Fig. 5: Limb use asymmetries in 6-OHDA treated rats.

As can be seen in Figure 6, there was a significant relationship between spontaneous limb use asymmetry scores and dopamine depletion, as determined by tyrosine hydroxylase levels, in 6-OHDA treated rats.

Fig. 6: Limb use asymmetries and dopamine depletion in 6-OHDA treated rats.

Forced overreliance on the impaired forelimb has previously been shown to exaggerate neuronal injury following electrolytic lesions of the cerebral cortex (Kozlowski et al., 1996). The results of the present study suggest that functional outcome following forced use of the impaired forelimb depends on the model being studied. If forced use follows focal cerebral ischemia, the volume of the infarct is increased and behavioral outcome worsened. In contrast, forced use appears to be benefical following a 6-OHDA dopamine lesion of the medial forebrain bundle (MFB), increasing dopamine levels in the striatum as compaired to lesioned animals not forced to use the impaired forelimb and dramatically decreasing behavioral deficits.

The difference in the effects of forced motor rehabilitation are likely due to differences between these two models of neural degeneration. Tandem occlusion of the left MCA combined with occlusion of the left CCA resulted in ischemic damage to the cerebral cortex. Forced motor therapy directed at the contralateral forelimb was begun after primary neuronal injury had already occurred, possibly exaggerating the cascade of neurodegenerative events leading to secondary injury, thus increasing infarct volume and worsening behavioral deficits. In contrast, injection of 6-OHDA into the MFB causes slow degeneration of dopamine terminals in the striatum. In this case, forced rehabilitation of the contralateral limb occurred during degeneration of the striatum, and may have preserved dopaminergic terminals, thus decreasing functional deficits that would ordinarily result from a 6-OHDA lesion.

Conclusions:

Forced motor therapy increases infarct volume following focal cerebral ischemia Forced motor therapy exaggerated post-ischemic behavioral deficits Dopaminergic terminals may be spared by forced rehabilitation Behavioral deficits that would ordinarily result from a 6-OHDA lesion are dramatically reduced by forced motor therapy