Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

Summary

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

Abstract

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

Introduction

Recent discoveries have demonstrated that the adult mammalian brain can undergo a remarkable degree of plasticity. Adult-born neurons are generated throughout adulthood in specialized niches of the mammalian brain¹. What is the function of these adult-born neurons? And more so, do they play a role in physiology and behavior? Studies on this topic have traditionally focused on the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus; however, recent studies have characterized neurogenesis in other brain regions such as the mammalian hypothalamus². Neurogenesis has been reported in the postnatal and adult hypoth....

Protocol

Animal Usage

Obtain approval from institutional Animal Care and Use Committee for standard care and use protocols. The current protocol was developed for focal irradiation studies on 5.5-10 week old adult C57BL6/J mice, as previously described (Figure 2)². However, other ages and small animal species (rats, hamsters, ground squirrels, etc.) can also be used, provided that effective anesthesia protocols and a radiographic reference atlas allowing identification of the region of interest (ROI) are available.

1. Calibration of CT-guided Radiological Platform

Results

Assessing CT-guided Targeting and Accuracy

The mechanical calibration of the system is critical for ensuring that beams from various angles all intersect in a single point. Calibration was accomplished with a semi-automatic imaging-based method, where end-to-end alignment accuracy has been measured to be 0.2 mm²⁹. This accuracy is highly critical as the volume of the hypothalamic median eminence structure is small². To test this calibration, we measured dose distributions wi.......

Discussion

CT-guided focal irradiation (CFIR) is a novel and complete system approach capable of delivering radiation fields to targets in small animals under robotic control using CT-guidance³². The capability of CFIR to deliver highly focused beams to small animal models provides new research opportunities to bridge laboratory research and clinical translation. This paper describes the CFIR approach for precise radiation delivery to specifically target a hypothalamic neural progenitor population. We demonstrate here ho.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SARRP research platform	Xstrahl	RS225A	http://www.xstrahl.com/xstrahlrs225.htm
SARRP irradiation bunker	Xstrahl		Optional, but radiation exposure should be contained with alternative lead shielding
GAF chromic film	IPS	GAFchromic ETB2
Mouse phantom	Gammex	457	Purchase 0.5 cm x 30 cm x 30 cm solid water slabs from Gammex and cut to desired size.
Mouse anti-phospho-histone H2AX Ser139 antibody	Millipore, Inc.	05-636	clone JBW301
High-fat rodent diet	Research Diets	D12492i	60% of the calories as fat, food should be irradiated
Isoflurane	Baxter Healthcare Corporation	10019-360-40
0.01 M Sodium citrate	Fisher Scientific		1.471 g of sodium citrate dissolved in 500 ml deionized water
Superfrost Plus slides	Fisher Scientific	12-550-15
DAPI	Fisher Scientific		nuclear counterstain
Mounting medium	Fisher Scientific		Vectashield or Gelvatol is preferred