In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Summary

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

Abstract

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient's pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [¹⁸F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

Introduction

The term neurostimulation encompasses a number of different techniques aimed at stimulating the nervous system with a therapeutic objective¹. Among them, deep brain stimulation (DBS) stands out as one of the most widespread neurostimulation strategies in clinical practice. DBS consists of the stimulation of deep brain nuclei with electrical pulses delivered by a neurostimulator, implanted directly into the patient's body, through electrodes placed into the brain target to be modulated by stereotactic surgery. The number of articles evaluating the feasibility of DBS application in different neurological and psychiatric disorders is continuously growing², although only some of them have been approved by the Food and Drug Association (FDA) (i.e., essential tremor, Parkinson's disease, dystonia, obsessive-compulsive disorder, and medically refractory epilepsy)³. Furthermore, a large number of brain targets and stimulation protocols are under research for DBS treatment of many more pathologies than officially approved, but none of them are considered definitive. These inconsistencies in DBS research and clinical procedures may in part be due to a lack of full understanding of its mechanism of action⁴. Therefore, huge efforts are being made to decipher the in vivo effects of DBS on brain dynamics, as every advance, however small, will help refine DBS protocols for greater therapeutic success.

In this context, molecular imaging techniques open a direct window to observe in vivo neuromodulatory effects of DBS. These approaches provide the opportunity not only to determine the impact of DBS while it is being applied but also to unravel the nature of its consequences, prevent undesired side effects and clinical improvement, and even adapt stimulation parameters to the patient's needs⁵. Among these methods, positron emission tomography (PET) using 2-deoxy-2-[¹⁸F]fluoro-D-glucose (FDG) is of particular interest because it provides specific and real-time information on the activation state of different brain regions⁶. Specifically, FDG-PET imaging provides an indirect evaluation of neural activation based on the physiological principle of metabolic coupling between neurons and glial cells⁶. In this sense, several clinical studies have reported DBS-modulated brain activity patterns using FDG-PET (see³ for review). Nevertheless, clinical studies easily incur several drawbacks when focusing on patients, such as heterogeneity or recruitment difficulties, which strongly limit their research potential⁶. This context leads researchers to use animal models of human conditions to evaluate biomedical approaches before their clinical translation or, if already applied in clinical practice, to explain the physiological origin of therapeutic benefits or side effects. Thus, despite the large distances between human pathology and the modeled condition in laboratory animals, these preclinical approaches are essential for a safe and effective transition into clinical practice.

This manuscript describes an experimental DBS protocol for murine models, combined with a longitudinal FDG-PET study, in order to assess the acute consequences of DBS on brain metabolism. The outcomes obtained with this protocol may help to unravel the intricate modulatory patterns induced on brain activity by DBS. Therefore, a suitable experimental strategy to examine in vivo the consequences of stimulation is provided, allowing clinicians to anticipate therapeutic effects under specific circumstances and then adapt stimulation parameters to the patient's needs.

Protocol

Experimental animal procedures were conducted according to the European Communities Council Directive 2010/63/EU, and approved by the Ethics Committee for Animal Experimentation of the Hospital Gregorio Marañón. A graphical summary of the experimental protocol is shown in Figure 1A. 1. Brain target localization by in vivo neuroimaging Animal preparation NOTE: Male Wistar rats of ~300 g were used. Place the …

Representative Results

The animals were sacrificed using CO2 at the end of the study or when the animal’s welfare was compromised. An example of a complete PET/CT study from an operated animal is shown in Figure 3. Thus, the electrode inserted into the rat brain can be clearly observed in the CT image shown in Figure 3A. This imaging modality provides good anatomical information and facilitates the registration of FDG-PET images, given that functional modalities …

Discussion

Given the advances in the understanding of brain function and the neural networks involved in the pathophysiology of neuropsychiatric disorders, more and more research is recognizing the potential of DBS in a wide range of neurologically-based pathologies². However, the mechanism of action of this therapy remains unclear. Several theories have attempted to explain the effects obtained in specific pathological and stimulation circumstances, but the heterogeneity of the proposed studies makes it ver…

Materials

7-Tesla Biospec 70/20 scanner	Bruker, Germany	SN0021	MRI scanner for small animal imaging
Betadine	Meda Pharma S.L., Spain	644625.6	Iodine solution (iodopovidone)
Beurer IL 11	Beurer	SN87318	Infra-red light
Bipolar cable 50 cm w/50 cm mesh covering up to 100 cm	Plastics One, USA	305-305 (CM)
Bipolar cable TT2  50 cm up to 100 cm	Plastics One, USA	305-340/2	Bipolar cable TT2  50 cm up to 100 cm
Buprex	Schering-Plough, S.A	961425	Buprenorphine (analgesic)
Ceftriaxona Reig Jofré 1g IM	Laboratorio Reig Jofré S.A., Spain	624239.1	Ceftriaxone (antibiotic)
Commutator	Plastics One, USA	SL2+2C	4 Channel Commutator for DBS
Concentric bipolar platinum-iridium electrodes	Plastics One, USA	MS303/8-AIU/Spc	Electrodes for DBS
Driller	Bosh	T58704	Driller
FDG	Curium Pharma Spain S.A., Spain	—–	2-[18F]fluoro-2-deoxy-D-glucose (PET radiotracer)
Heating pad	DAGA, Spain	23115	Heating pad
Ketolar	Pfizer S.L., Spain	776211.9	Ketamine (anesthetic drug)
Lipolasic 2 mg/g	Bausch & Lomb S.A, Spain	65277	Ophthalmic lubricating gel
MatLab R2021a	The MathWorks, Inc		Support software for SPM12
MRIcro	McCausland Center for Brain Imaging,  University of South Carolina, USA	v2.1.58-0	Software for imaging preprocessing and analysis
Multimodality Workstation (MMWKS)	BiiG, Spain		Software for imaging processing and analysis
Omicrom VISION VET	RGB Medical Devices, Spain	731100 ReV B	Cardiorrespiratory monitor for small imaging
Prevex Cotton buds	Prevex, Finland	—–	Cotton buds
Sevorane	AbbVie Spain, S.L.U, Spain	673186.4	Sevoflurane (inhalatory anesthesia)
Small screws	Max Witte GmbH	1,2 x 2 DIN 84 A2	Small screws
Standard U-Frame Stereotaxic Instrument for Rat, 18° Ear Bar	Harvard Apparatus, USA	75-1801	Two-arms Stereotactic frame for rat
Statistical Parametric Mapping (SPM12)	The Wellcome Center for Human Neuroimaging, UCL Queen Square Institute of Neurology, UK	SPM12	Software for voxel-wise imaging analysis
STG1004	Multi Channel Systems GmbH, Germany	STG1004	Isolated stimulator
SuperArgus PET/CT scanner	Sedecal, Spain	S0026403	NanoPET/CT scanner for small animal imaging
Suture thread with needle, 1/º	Lorca Marín S.A., Spain	55325	Braided natural silk non-absorbable suture 1/0, with triangle needle
Technovit 4004 (powder and liquid)	Kulzer Technique, Germany	64708471; 64708474	Acrylic dental cement for craniotomy tap
Wistar rats (Rattus norvergicus)	Charles River, Spain	animal facility	Animal model used
Xylagesic	Laboratorios Karizoo, A.A, Spain	572599-4	Xylazine (anesthetic drug)
	Normon S.A., Spain	602910	Mepivacaine in gel for topical use