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

Deep brain stimulation (DBS) is the most common neurosurgical treatment for Parkinson\u27s 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

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain

Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson\u27s disease. Although viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be because of the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing Parkinson\u27s disease and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared with the young adult. We examined recombinant adeno-associated virus 2/5-mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra aged rats displayed 40%-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Furthermore, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP messenger RNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less messenger RNA and protein expression

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

Striatal pleiotrophin overexpression provides functional and morphological neuroprotection in the 6-hydroxydopamine model

Neurotrophic factors are integrally involved in the development of the nigrostriatal system and in combination with gene therapy, possess great therapeutic potential for Parkinson\u27s disease (PD). Pleiotrophin (PTN) is involved in the development, maintenance, and repair of the nigrostriatal dopamine (DA) system. The present study examined the ability of striatal PTN overexpression, delivered via psueudotyped recombinant adeno-associated virus type 2/1 (rAAV2/1), to provide neuroprotection and functional restoration from 6-hydroxydopamine (6-OHDA). Striatal PTN overexpression led to significant neuroprotection of tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc) and THir neurite density in the striatum, with long-term PTN overexpression producing recovery from 6-OHDA-induced deficits in contralateral forelimb use. Transduced striatal PTN levels were increased threefold compared to adult striatal PTN expression and approximated peak endogenous developmental levels (P1). rAAV2/1 vector exclusively transduced neurons within the striatum and SNpc with approximately half the total striatal volume routinely transduced using our injection parameters. Our results indicate that striatal PTN overexpression can provide neuroprotection for the 6-OHDA lesioned nigrostriatal system based upon morphological and functional measures and that striatal PTN levels similar in magnitude to those expressed in the striatum during development are sufficient to provide neuroprotection from Parkinsonian insult. © The American Society of Gene & Cell Therapy

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

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

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

Statistical Table.

<p>Statistical Table.</p

Striatal Pleiotrophin Overexpression Provides Functional and Morphological Neuroprotection in the 6-Hydroxydopamine Model

Neurotrophic factors are integrally involved in the development of the nigrostriatal system and in combination with gene therapy, possess great therapeutic potential for Parkinson's disease (PD). Pleiotrophin (PTN) is involved in the development, maintenance, and repair of the nigrostriatal dopamine (DA) system. The present study examined the ability of striatal PTN overexpression, delivered via psueudotyped recombinant adeno-associated virus type 2/1 (rAAV2/1), to provide neuroprotection and functional restoration from 6-hydroxydopamine (6-OHDA). Striatal PTN overexpression led to significant neuroprotection of tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc) and THir neurite density in the striatum, with long-term PTN overexpression producing recovery from 6-OHDA-induced deficits in contralateral forelimb use. Transduced striatal PTN levels were increased threefold compared to adult striatal PTN expression and approximated peak endogenous developmental levels (P1). rAAV2/1 vector exclusively transduced neurons within the striatum and SNpc with approximately half the total striatal volume routinely transduced using our injection parameters. Our results indicate that striatal PTN overexpression can provide neuroprotection for the 6-OHDA lesioned nigrostriatal system based upon morphological and functional measures and that striatal PTN levels similar in magnitude to those expressed in the striatum during development are sufficient to provide neuroprotection from Parkinsonian insult