SHH knockdown increases microglial activity in the host brain.

<p>Host microglia expressing cd11b and cd68 antigens (expressed by activated microglia) were analyzed two weeks after NPC implantation. A greater number of both cd68⁺ and cd11b⁺ cells were noted in animals that had received the SHH or GDNF+SHH silenced NPCs (D-F, J-L, P-R, V-X high mag images in b, d, f, g) in comparison to rats with control (A-C, M-O; high mag images in a and e) or GDNF silenced (G-I, S-U; high mag images in c and g) grafts. The number of cd11b⁺ and cd68⁺ cells present in regions adjacent to the transplanted NPCs were quantified using confocal microscopy, and it was confirmed that the decrement of SHH in grafted NPCs had in fact resulted in a significant (p<0.001) activation of microglia in the host neural environment (Y, Z). Values are expressed as mean ± SEM [***p<0.001 compared to control, one way ANOVA with Bonferroni’s post test]. Scale bar: A-S—50 μm, a-f—15 μm.</p

<i>In vitro</i> lentiviral silencing of GDNF and SHH in NPCs.

<p>Western blotting analyses of cultured NPCs indicated that the cells expressed only GDNF (~25kda) and SHH (~45kda), but no detectable SDF1α (~11kda), under basal conditions (E-G; GDNF and SHH were run on the same gel and membrane divided, whereas SDF1 was run on a separate gel). Therefore, an FIV based <i>in vitro</i> RNA interference approach was used to knock down the expression of GDNF, SHH or both in donor NPCs before transplantation (B). A schematic diagram of pVETL construct used for expression of shRNAs and the GFP reporter is depicted in (A). After testing, the two most efficient shRNA constructs targeting GDNF or SHH were cloned into a FIV eGFP vector and subsequently infected into the NPCs in culture (D, shows a representative image of <i>in vitro</i> NPCs infected with control eGFP vector). The NPCs did not show a significant change in survival and proliferative ability after the shRNA infections (H, I). When the silencing efficiency of the virally infected shRNA’s was examined through mRNA (qPCR; J, K) and protein (western blot; L-O) analyses, it was determined that in particular the GDNF1 and SHH4 constructs were able to most significantly (p<0.001) inhibit GDNF and SHH expression respectively, when compared to mock (eGFP vector) and scrambled (non-targeted shRNA) controls, and were focused upon in the rest of the study. Image E displays control FIV eGFP expressing NPCs <i>in vivo</i>, 2 weeks after transplantation. Values are expressed as mean ± SEM [*p<0.05, **p<0.01, ***p<0.001 compared to scrambled control, one-way ANOVA with Tukey’s post hoc test]. Scale bars: A, B, C (in C)– 25 μm; D, E—100 μm.</p

Effect of SHH and GDNF silencing on the number of grafted NPCs.

<p>We assessed the number of GFP expressing donor cells (A) in both the striatum and nigra of rats receiving control and silenced NPCs. Estimates of grafted GFP⁺ cells in hematoxylin counterstained tissue sections (B) showed no significant differences in the striatum or SN of SHH or GDNF silenced animals when compared to controls. However the animals receiving GDNF+SHH silenced grafts showed reduced numbers of grafted cells in the striatum in comparison to animals with control cells (C). Values are expressed as mean ± SEM [*p<0.05, **p<0.01, ***p<0.001 compared to shC, two way ANOVA with Bonferroni’s post test].Scale bars: A—25 μm; B—10 μm.</p

Inhibition of SHH influences the phenotypic fate of grafted NPCs.

<p>We analyzed the co-expression of antigens specific to nestin (undifferentiated NPCs, A-C), immature neurons (tuj1, D-F), astrocytes [(s100β (G-I) and oligodendroglia [RIP (J-L)] in the GFP NPCs. Counts of grafted GFP cells indicated that while the number of nestin⁺ cells was significantly lower, compared to control NPCs in donor cells silenced for SHH or GDNF and SHH, shGDNF grafts did not show such an effect (a). This was true for grafts both in the striatum as well as SN. Representative images of nestin stained grafts are shown in M, Q, U. The GDNF silenced grafts on the other hand showed a significant (p<0.001) reduction in the number of cells expressing the neuron marker tuj1 (b). Such a decline in tuj1 expression was not observed in the SHH silenced NPCs. Representative images of tuj1 stained grafts are shown in O, S, W. Further, the number of S100β⁺ cells was greater in striatal grafts with SHH knock-down, with no such influence observed in any of the other experimental groups (c). Representative images of S100β⁺ cells in grafts are shown in N, R, V. Finally, with respect to oligodendrocyte differentiation, no significant differences were observed between control and silenced NPCs (d). Representative images of RIP staining in grafts are shown in P, T, X. Values are expressed as mean ± SEM [*p<0.05, **p<0.01, ***p<0.001 compared to shC, one way ANOVA with Bonferroni’s post test]. Scale bars: A-L—10 μm; M-X—30 μm.</p

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

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