High-Frequency Stimulation of the Subthalamic Nucleus Counteracts Cortical Expression of Major Histocompatibility Complex Genes in a Rat Model of Parkinson’s Disease

<div><p>High-frequency stimulation of the subthalamic nucleus (STN-HFS) is widely used as therapeutic intervention in patients suffering from advanced Parkinson’s disease. STN-HFS exerts a powerful modulatory effect on cortical motor control by orthodromic modulation of basal ganglia outflow and via antidromic activation of corticofugal fibers. However, STN-HFS-induced changes of the sensorimotor cortex are hitherto unexplored. To address this question at a genomic level, we performed mRNA expression analyses using Affymetrix microarray gene chips and real-time RT-PCR in sensorimotor cortex of parkinsonian and control rats following STN-HFS. Experimental parkinsonism was induced in Brown Norway rats by bilateral nigral injections of 6-hydroxydopamine and was assessed histologically, behaviorally, and electrophysiologically. We applied prolonged (23h) unilateral STN-HFS in awake and freely moving animals, with the non-stimulated hemisphere serving as an internal control for gene expression analyses. Gene enrichment analysis revealed strongest regulation in major histocompatibility complex (MHC) related genes. STN-HFS led to a cortical downregulation of several MHC class II (RT1-Da, Db1, Ba, and Cd74) and MHC class I (RT1CE) encoding genes. The same set of genes showed increased expression levels in a comparison addressing the effect of 6-hydroxydopamine lesioning. Hence, our data suggest the possible association of altered microglial activity and synaptic transmission by STN-HFS within the sensorimotor cortex of 6-hydroxydopamine treated rats.</p></div

Stereology of tyrosine-hydroxylase positive midbrain neurons.

<p>Representative sections (2,5x lens) of the midbrain depicting the rootlets of the third oculomotor cranial nerve (3n), VTA, SNc and SNr stained for tyrosine-hydroxylase (TH) of an example control (A) and example PD rat (D). Photomicrographs show magnifications taken with a 40x lens and depict TH-positive dopaminergic cells in the SNc (B) and VTA (C) of a vehicle injected rat, as well as of a 6-OHDA injected rat (E and F, respectively). Results of unbiased stereological cell counting (G) are presented as estimated cell density (mean±SD cells/mm³) for the VTA and SNc of all rats included in gene expression profiling (n = 3 PD, n = 3 control). Filled black bars indicate cell estimates respectively of vehicle and gray filled bars of 6-OHDA injected rats. Statistically significant differences are marked with an asterisk. 3n, 3rd oculomotor cranial nerve; VTA, ventral tegmental area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; TH, tyrosine-hydroxylase; 6-OHDA, 6-hydroxydopamine.</p

Electrode placement and tissue reaction.

<p>Representative traces (A) of microelectrode guided targeting of the STN under ketamine/xylazine anesthesia. Note that the STN trace is characterized by elevated background activity, bursts and irregular high-frequency firing. A representative electrode trajectory (B) targeting the STN (cresyl-violet staining, arrowheads indicate trajectory, scale bar 500 μm). The STN, cerebral peduncle (cp) and optic tract (opt) are circumscribed with black surrounding lines. Photomicrographs of the STN at 20x- and 40x-magnification (scale bars 100 μm and 50 μm, respectively) reveal mild reactive tissue infiltration with mononuclear cells at the electrode tip. Bars (C) indicate the area (mean±SD) infiltrated by mononuclear cells at the tip of stimulated (HFS, black bar) and non-stimulated electrodes (no HFS, white bar; n = 6 in each group). STN, subthalamic nucleus; HFS, high-frequency stimulation; cp, cerebral peduncle; opt, optic tract.</p

Real-time RT-PCR.

<p>Real-time RT-PCR results for MHC class II genes Cd74 (A) and RT1-Da (B) regulated by unilateral STN-HFS. Lesion effect comparison (C) for both MHC class II genes. Filled black bars indicate signal-log changes of RT-PCR analysis and open bars the results of microarray experiments (mean±SD). Asterisks denote statistically significant results. MHC, major histocompatibility complex; STN, subthalamic nucleus; HFS, high-frequency stimulation.</p

Spontaneous locomotor activity revealed parkinsonian phenotype.

<p>The upper row (A) depicts spontaneous 23h locomotor activity of a representative control rat, the lower row (B) of an example PD rat. Recordings were performed before (left) and after 6-OHDA or vehicle injections (left-middle) as well as before (right-middle) and during unilateral STN-HFS (right). The lower row depicts descriptive statistics of spatial spread (C) and maximal movement speed derivative (max-SD; D) averaged over episodes of full blown motion for all four recording times (see above). Markers and error-bars indicate the mean±SD of animals included in gene expression profiling (n = 3 each group; controls filled-black, PD rats open-gray symbols). Thin lines indicate each single animal included in gene expression profiling. PD, Parkinsońs disease; 6-OHDA, 6-hydroxydopamine; STN, subthalamic nucleus; HFS, high-frequency stimulation.</p

Flowchart of the experimental design.

<p>The sequence of experimental steps (i.e., 6-OHDA or vehicle injections, pre- and post-operative locomotion monitoring, STN-HFS electrode implantation, awake LFP recordings, pre-HFS locomotion monitoring, awake STN-HFS under locomotion monitoring, tissue isolation), number of included animals and time between steps is given in a flowchart. 6-OHDA, 6-hydroxydopamine; STN, subthalamic nucleus, HFS, high-frequency stimulation; LFP, local field potential.</p

Spectral analysis of cortical and subthalamic local field potentials.

<p>Representative examples (A) of oscillatory activity in the raw local field potentials (LFP) recorded from STN (upper row) and frontal ECoG (lower row) of controls (left column) and 6-OHDA treated parkinsonian rats (right column). Time-frequency plots (B) of spectral power during 180 s of quiet rest from all PD animals included in gene expression analyses. Power spectra (C) calculated from frontal ECoG and STN-LFP data of parkinsonian rats (light gray lines/shadings) and controls (dark gray lines/shadings): (i) low-frequency range (1–45 Hz) and (ii) high-frequency range (45–80 Hz) of ECoG-power spectra pooled across all hemispheres (n = 6). (iii) Low-frequency power spectrum of STN-LFP ipsilateral to the side of HFS and (iv) contralateral to the side of HFS (n = 3 each). (v) High-frequency power spectrum of STN-LFP pooled across all hemispheres. Asterisks indicate significantly different frequency bands. Note the dampening at 50 Hz that resulted from bandpass filtering of line noise. LFP, local field potential; STN, subthalamic nucleus; ECoG, electrocorticogram; 6-OHDA, 6-hydroxydopamine, HFS, high-frequency stimulation.</p

STN-HFS regulated MHC genes in sensorimotor cortex.

<p>List of all microarray regulated genes exceeding the slr-threshold of ±0.6 and showing homonymous regulation in both intra- and across-subject comparisons. Gene name, gene symbol and Affymetrix ID are given with the respective decrease (D) or increase (I) count as well as the mean slr. Data are presented for 6-OHDA and vehicle treated rats and for the effect of lesion analysis that compared the gene expression change between the non-stimulated hemispheres of 6-OHDA and control rats.</p><p>Slr, signal-log ratio; 6-OHDA, 6-hydroxydopamine.</p

CTC grade of SAE vs relatedness to DBS therapy.

<p>CTC grade of SAE vs relatedness to DBS therapy.</p

Sum of AEs defined by same severity, reversibility, and attribution to DBS therapy.

<p>Green, reversible; orange, non reversible; grey, unknown. The actual number of AEs is presented. The dotted area indicates AEs that were <i>severe</i> or worse and at least <i>possibly</i> related to DBS therapy and, thus, regarded the most critical. N.B. The number of affected patients may be less than the number indicated because individual patients may have suffered from more than one AE of respective groups (e.g. impairment of gait and speech rated as <i>mild</i>, <i>probably</i> related and <i>non-reversible</i>).</p