High-Frequency Stimulation of the Rat Entopeduncular Nucleus Does Not Provide Functional or Morphological Neuroprotection from 6-Hydroxydopamine

<div><p>Deep brain stimulation (DBS) is the most common neurosurgical treatment for Parkinson’s disease (PD). Whereas the globus pallidus interna (GPi) has been less commonly targeted than the subthalamic nucleus (STN), a recent clinical trial suggests that GPi DBS may provide better outcomes for patients with psychiatric comorbidities. Several laboratories have demonstrated that DBS of the STN provides neuroprotection of substantia nigra pars compacta (SNpc) dopamine neurons in preclinical neurotoxin models of PD and increases brain-derived neurotrophic factor (BDNF). However, whether DBS of the entopeduncular nucleus (EP), the homologous structure to the GPi in the rat, has similar neuroprotective potential in preclinical models has not been investigated. We investigated the impact of EP DBS on forelimb use asymmetry and SNpc degeneration induced by 6-hydroxydopamine (6-OHDA) and on BDNF levels. EP DBS in male rats received unilateral, intrastriatal 6-OHDA and ACTIVE or INACTIVE stimulation continuously for two weeks. Outcome measures included quantification of contralateral forelimb use, stereological assessment of SNpc neurons and BDNF levels. EP DBS 1) did not ameliorate forelimb impairments induced by 6-OHDA, 2) did not provide neuroprotection for SNpc neurons and 3) did not significantly increase BDNF levels in any of the structures examined. These results are in sharp contrast to the functional improvement, neuroprotection and BDNF-enhancing effects of STN DBS under identical experimental parameters in the rat. The lack of functional response to EP DBS suggests that stimulation of the rat EP may not represent an accurate model of clinical GPi stimulation.</p></div

Comparison of Primate GPi, Rat EP, Primate SNpr and Rat SNpr.

<p>GPi = globus pallidus interna, EP = entopeduncular nucleus, SNpr = substantia nigra pars reticulata, GABA = γ-aminobutyric acid, STN = subthalamic nucleus, GPe = globus pallidus externa, PPN = pedunculopontine tegmental nucleus</p><p>Comparison of Primate GPi, Rat EP, Primate SNpr and Rat SNpr.</p

Experimental overview for EP DBS.

<p><i>Experiment 1</i>. On Day 1, rats received an electrode implanted in the EP. After three weeks of recovery, rats were randomly assigned to ACTIVE or INACTIVE stimulation for a two-week interval. Rats tolerated stimulation of the EP for two weeks as they otherwise would for STN DBS for the same duration. Rats were sacrificed and perfused on Day 36. <i>Experiment 2</i>. On Day 0, rats were assessed for baseline forelimb asymmetry using the cylinder task. On Day 1, rats received unilateral, intrastriatal 6-OHDA and an electrode was implanted during the same surgical session in the EP ipsilateral to the lesion. After two weeks of nigrostriatal degeneration (≈50% loss of SNpc neurons, as determined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133957#pone.0133957.ref020" target="_blank">20</a>]), rats were reassessed for forelimb asymmetry, and rats with sufficient deficits in contralateral paw use were randomly assigned to receive ACTIVE or INACTIVE stimulation for a two-week interval. On Day 28, rats were reassessed using the cylinder task (“Stim On” condition), and after a twenty-four-hour washout after the cessation of stimulation, the rats were again assessed using the cylinder task (“Stim Off” condition). Rats were sacrificed and perfused on Day 30.</p

Electrodes implanted in the EP remain in position over the two-week stimulation interval.

<p>Representative photomicrographs illustrate unilateral electrode placement in the EP following Kluver-Barrera staining. (<b>A</b>) Low magnification image shows the approximate placement of the stimulating electrode prior to its removal post mortem and the tissue damage related to the removal process. The active electrode tip diameter is 150 μm whereas the shaft of the electrode is 400 μm in diameter. (<b>B</b>) High magnification of the electrode tip’s position in the EP. <b>(C)</b> EP neurons are visible in a nearby coronal section (≈160 μm caudal), indicating that a significant portion of the EP remained intact. Rats in which electrodes were found to be positioned more than 250 μm away from the EP were excluded from analysis based on previous estimates of current spread [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133957#pone.0133957.ref020" target="_blank">20</a>]. Scale bar in A = 1000 μm, C = 500 μm.</p

EP DBS does not increase BDNF.

<p>BDNF protein levels were normalized to total protein in key basal ganglia structures of intact rats after a two-week stimulation interval. Data from each structure were normalized to the corresponding structure from the INACTIVE, contralateral (to 6-OHDA and electrode lead) hemisphere to control for the potential effect of dopamine denervation or electrode implantation on BDNF levels. Samples were obtained for the ipsilateral (Ipsi) and contralateral (Contra) substantia nigra (SN), striatum (STR), primary motor cortex (M1), thalamus and hippocampus. No significant difference was observed between ACTIVE and INACTIVE stimulation groups nor within animals between sides, though there was a trend toward significance between the Active and Inactive SN bilaterally.</p

Statistical Table.

<p>Statistical Table.</p