Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Summary

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Abstract

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus¹. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

Introduction

The history behind the development of Deep Brain Stimulation as a neurosurgical technique dates back to the 1870s when the possibility of electrically stimulating the brain circuitry was explored². The use of chronic high-frequency stimulation as treatment for neuronal disorders started in the 1960s³. Later in the 1990s with the advent of chronic implantation DBS electrodes^4-6, the number of neuronal disorders that were treated by DBS continued to increase. Deep Brain Stimulation was first used in the United States as a treatment for essential tremor⁶. Today the surgery is used widely to treat neuronal disorders that are cur....

Protocol

NOTE: Ethics Statement: All procedures discussed in this manuscript are in accordance with the NIH guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals) and are approved by the Harvard Medical School IACUC Committee.

1. Pre-surgical Preparation

1. Make sure that all surgical instruments are sterilized by either autoclaving or by cleansing with antiseptic solution and/or ethanol as necessary. Where possible, use sterile disposable equipment such as scalpels, needle and syringes.
2. Cover the workbench with surgical drapes and ensure that there is a biohazard waste disposal available.
3. Weig....

Results

Figures 1A and 1B show the relative expression of BDNF and GABRD relative to the control gene β-actin. BDNF, a neurotrophin is often associated with neuroprotective effects in many neuronal diseases^38-41. It is therefore interesting to analyze the expression profile of BDNF in response to stimulation of the ATN which yields therapeutic benefits to epileptic patients. In Figure 1A which shows the gene expression profile of BDNF across the indicated time-points .......

Discussion

Following the landmark work by Benabid et al. in using deep brain stimulation to treat Parkinson’s disease and essential tremor, the DBS surgical technique has been investigated with much interest over the past decade to treat many neurological disorders^6,10,43. DBS studies targeting various neuro-anatomical regions of the brain circuitry are currently performed by many groups to address major neuronal diseases and are in various stages of clinical trials. Stimulation of the subthalamic nucleus .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Deep Brain Stimulation Surgery
Stereotactic frame	Kopf Instruments	Model 900
Drill	Dremmel	7700, 7.2 V
Scalpel	BD	372610
Ketamine	Patterson Veterinary	07-803-6637	Schedule III Controlled Substance, procurement, use and storage according to institutional rules
Xylazine	Patterson Veterinary	07-808-1947
Buprenorphine	Patterson Veterinary	07-850-2280	Schedule III Controlled Substance, procurement, use and storage according to institutional rules
Surgical staples	ConMed Corporation	8035
Sutures (3-0)	Harvard Apparatus	72-3333
Syringe (1 ml, 29 1/2 G)	BD	329464	Sterile, use for Anesthesia administration intraperitoneally
Syringe (3 ml, 25 G)	BD	309570	Sterile, use for Analgesia administration subcutaneously
Needles	BD	305761	Sterile, use for clearing broken bone pieces from the burr holes
Ethanol	Fisher Scientific	S25309B	Use for general sterilization
Eye Lubricant	Fisher Scientific	19-898-350
Stimulator	Medtronic	Model 3628
DBS electrodes	Rhodes Medical Instruments, CA	SNEX100x-100 mm	Electrodes are platinum, concentric and bipolar
Betadine (Povidone-Iodine)	PDI	S23125	Single use swabsticks, use for sterilizing the scalp before making incision
Brain Dissection and Hippocampal tissue isolation
Acrylic Rodent Brain Matrix	Electron Microscopy Sciences	175-300	www.emsdiasum.com
Razor Blade	V W R	55411-050
Guillotine Scissors	Clauss	18039	For decapitation, make sure these scissors are maintained in clean and working condition
Scissors	Codman Classic	34-4098	Use for removing the brain from the skull
Forceps	Electron Microscopy Sciences	72957-06	Use for removing the brain from the skull and for handling during dissection
Phosphate Buffered Saline	Boston Bioproducts	BM-220
RNA Extraction and cDNA Preparation
Tri Reagent	Sigma	T9424	Always use in a fume hood and wear protective goggles while handling; avoid contact with skin
Syringe (3 ml, 25 G)	BD	309570	Use for tissue homogenization
Chloroform	Fisher Scientific	BP1145-1	Always use in a fume hood and wear protective goggles while handling; avoid contact with skin
Isopropanol	Fisher Scientific	A416-1
Glycogen	Thermo Scientific	R0561
Dnase I Kit	Ambion	AM1906
Superscript First Strand Synthesis Kit	Invitrogen	11904-018
Tabletop Microcentrifuge	Eppendorf	5415D
Quantitative PCR
SYBR Green PCR Kit	Qiagen	204143
Custom Oligos	Invitrogen	10668051
PCR Plates (96 wells)	Denville Scientific	C18080-10
Optical Adhesive Sheets	Thermo Scientific	AB1170
Nuclease free Water	Thermo Scientific	SH30538-02
Real Time PCR Machine	Applied Biosystems	7500