Dynamic Cerebral Microdialysis during Pallidotomy and Thalamotomy in Parkinson’s Disease: A Preliminary Neurochemical Study

In Parkinson’s disease (PD), dopaminergic neurons reduce the regulation of glutamatergic (glutamate-Glu) input from the cortex to neostriatum (caudate and putamen nuclei) consequently leading to a hyperactivity of globus pallidus internae (GPi) neurons that release gamma-amino-butyric acid (GABA) into the thalamic ventrolateral (VL) nucleus. The objective of the present ex-periment was to measure changes in GABA and Glu in the caudate and the thalamus of 2 patients during the application of electrical stimuli following either a pallidotomy or a thalamotomy. Proper insertion of the electrode was tested by applying high frequency electrical pulses (HFEP). During these procedures, we obtained neurochemical information placing cerebral (CMD) microdialysis probes in caudate nucleus and VL nucleus of ipsi- and con-tra-lateral thalamus. In VL thalamus, extracellular GABA decreased during HFEP, tending to reach previous levels once HFEP was finalized. Following the pallido- or thalamotomy GABA decreased again. Similarly, in the contralateral VL thalamus, extracellular GABA levels showed a similar but less pro-nounced profile but did not show any decrement after the lesion. Caudate Glu decreases when HFEP is applied to the GPi and recovers to previous levels after HFEP, but did not decrease again after lesion (GPi-tomy), instead it continued to rise. These results suggest that HFEP exerts a similar but re-versible biochemical effect as thermopallido- or thermothalamotomy on GABA extracellular concentration in the ipsilateral VL thalamus. We also observe a distant effect of HFEP, but not of thermolesion, on contralateral thalamic GABA and ipsilateral caudate Glu.


Introduction
Parkinson's disease (PD) affects between 0.4% and 0.6% of the Venezuelan population (morbidity of Ministry of Health, 2013 and 2017).
Surgical therapies for PD include ablative procedures as pallidotomy [1] [2] and thalamotomy [3] and more conservative surgery as deep brain stimulation (DBS) [4] [5], although we prefer the term of deep brain electrical modulation (DBEM). Application of electrical pulses was used in the past to verify the correct placement of instruments in awake patients during surgery [6]. Generally, low frequency (<8 Hz) electrical pulses induced neural excitation and exacerbated symptoms or signs corresponding to the targeted region, whereas high frequency (>100 Hz) electrical pulses mimicked the effect of lesion and suppressed these symptoms and signs [6] [7]. The mechanisms responsible for this phenomenon are not clear. Some hypotheses include: 1) high frequency electrical pulses (HFEP) may silence the activity of neurons in the globus pallidum internae (GPi) or in the subthalamic nucleus (STN) neurons by a "depolarization block" [8] [9]; 2) HFEP may reshape the abnormal pattern of activity of the basal ganglia output nuclei [10] [11].
A pathophysiological hypothesis of the basal ganglia that attempted to explain PD and dyskinesias was suggested two decades ago by De Long [12] [13] [14].
The circuitry is schematically illustrated in Figure 1. For the purposes of this      Coordinates of the caudate nucleus are calculated referring to the center of the  image of the head of this nucleus, seen in the computed tomography, at the same plane of Vim nucleus.

Surgical Procedures
Microdialysis probes were perfused with a sterile solution (135 mM NaCl, 3.7 mM KCl, 1.0 mM MgCl 2 , 1.2 mM CaCl 2 and 10 mM NaHCO 3 , pH 7.4) at a flow rate of 1 microL/min in order to obtain basal levels. Sampling was started 30-min following the probe implantation, which is the time considered necessary to equilibrate extracellular neurotransmitter post-insertion.
Microdialysates were collected every 5 min. Sampling consisted of 2 basal samples, 1 sample during HFEP, 2 samples post-HFEP and 2 final samples following radiofrequency coagulation. Once basal samples were collected, electrodes were then advanced until target region (GPi or Vim) was reached. Impedance was measured during electrode advance and HFEP was applied (1 -4 volt, 110 Hz) to confirm electrode position once the target was reached and 1 sample was taken at this moment as previously noted. When the position of electrodes was definitive and after two 5-min samples were taken post-HFEP, a thermolesion was performed, 75˚C for 60 min, and 2 final samples were taken.

Sample Preparation and Analysis
Standard derivatization of samples was obtained with optimal FITC/GABA relation of GABA [24], and FITC/Glu relation for Glu (17,19), as previously published.
A Meridialysis CZE ® system (model 3ZPO, Mérida, Venezuela) equipped with an argon laser was used for measurement of samples, using the capillary elec-  (Figure 4). Results are expressed in percentage, related to the mean of the first 2 (basal) samples taken as 100%.

Results
In patient 1 (left Gpi-tomy) right dyskinesia disappeared progressively in the following weeks, and remained absent with a follow-up of 3 years. Patient 2 (Vim-tomy) had a transient right hemiparesis, probably due to peri-coagulation edema since it only lasted 1 week post-operatively. She immediately showed cessation of tremor at surgery, however, a mild tremor reappeared 6 months later, which could be controlled by medication.
GABA levels were measured in the ventro-lateral (VL) thalamus in both sides and Glu levels were measured in the left caudate nucleus in both patients.

GABA Levels in Ipsilateral Thalamus
In patient 1 (GPi-tomy) when HFEP was applied in Gpi, extracellular levels of GABA in VL-thalamus (GABA-VL) decreased and tended to regain previous levels when HFEP were halted. A similar profile of GABA-VL was observed in patient 2 (Vim-tomy) when HFEP were applied to the ipsilateral Vim. But the recuperation of the previous GABA-VL levels was slower when HFEP stopped in this patient ( Figure 5). After thermolesion, both patients showed a decrement of GABA-VL levels.

GABA Levels in Contralateral Thalamus
In contralateral VL, extracellular GABA-VL levels showed a similar but less marked profile, i.e. diminution of GABA levels, although less abruptly, during HFEP, and regained previous levels after HFEP stopped; but did not show any decrement after lesion. In fact, after lesion, GABA levels continue to rise. It  seems that HFEP indirectly affects contralateral thalamus, but not the thermolesion ( Figure 6).

Glu Levels in Ipsilateral Caudate Nucleus
Glu levels were measured in the left (ipsilateral) caudate nucleus (Figure 7). Glu

Discussions
According to the accepted model of the basal ganglia pathophysiology [12] [13] [14], we found that, indeed, VL-thalamic GABA levels are higher than after the surgical correction, as it would be expected. We cannot be sure if levels were higher than normal, since we lack any control of thalamic GABA levels. Howev-   The mechanism by which HFEP exerts an inhibitory effect is not well known.
Many hypotheses have arisen and remain to be verified. It has been proposed that inhibitory neurotransmitters are also released along with excitatory neurotransmitters resulting in an inhibitory final effect [30]. HFEP could also override abnormally patterned activity instead of inhibition [30]. This can be interpreted as if the neurons start firing in a frequency that disturbs normal depolarization, thus leading to "no depolarization" or "depolarization block" [8] [9], and thus avoiding any transmission of signal. Recently, it was suggested that globus pallidus neurons are normally desynchronized and their activity becomes synchronized in Parkinson's disease, thus HFEP may induce chaotic desynchronization by interacting with the intrinsic oscillatory mechanism of globus pallidus neurons [31]. These authors suggest a mechanism of action of HFEP in which synchrony, rather than firing rate, is the critical pathological feature. This might partly explain the HFEP mechanism.
Direct neurochemical demonstration of the hypothetical physiopathology of the basal ganglia in humans was not published before. These observations reinforce this hypothetical model. We did not find any previous report of CMD performed in surgery, during application of stimuli, i.e., or during electrophysiological tests. We call this "dynamic microdialysis".
This study can help in assessing the relevance of CMD. Further work, on a longer series, could give evidence to justify CMD as a tool in neurosurgery that can aid in precise diagnosis, thus guiding better functional neurosurgical interventions, and a mean to better understand the pathophysiology in Parkinson's disease.

Note
Our experience in stereotactic functional neurosurgery began in the Hospital Vargas in the late 60s, after neurosurgical training in Professor Lars Leksell's