Method Article

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

DOI:

10.3791/50716

⸱

November 14th, 2013

 ,  ,  ,  ,  , 
1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine

In This Article

Summary

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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

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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

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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 hypothalamus2-10, and the function of these newborn hypothalamic neurons remains an active area of investigation.

The functional characterization of adult-born neurons remains a significant challenge for the neuroscience field in general. Selective inhibition of specific neural progenitor populations remains limited by the lack of available molecular markers that are unique to single neural progenitor populations11. Thus, selective deletion of adult-born neurons from these neural progenitors via genetic targeting remains difficult. Likewise, viral delivery to target adult-born neurons suffers from potential confounding variables such as introducing injury and inflammation into the environment12.

New radiological tools are emerging, however, that allow one to circumvent these confounds and investigate these questions through accurate and precise anatomical targeting in small animals. Ionizing radiation inhibits the birth and differentiation of new neurons, and allows a noninvasive method to target neural progenitor populations13-15. Recently, we described a germinal region of the mammalian hypothalamic median eminence (ME) that we termed the hypothalamic proliferative zone (HPZ)2. We found that when young adult female mice were given a high-fat diet (HFD), levels of neurogenesis in HFD-fed mice were substantially higher than their normal chow (NC) fed controls in this ME region2. To test whether adult neurogenesis within the hypothalamic ME regulates metabolism and weight, we sought to disrupt this process. The median eminence is a small unilateral structure at the base of the third ventricle from which regulatory hormones are released. In order to inhibit proliferation and subsequent neurogenesis without altering the other physiological functions of this brain region, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of newly born adult neurons in the hypothalamic median eminence2.

A number of groups have employed radiation to suppress neurogenesis in canonical regions14-28. However, previous radiological approaches have generally targeted large areas, or often unintentionally also targeted multiple brain areas where neurogenesis has been reported, making it difficult to unambiguously associate any behavioral defects observed with defects in specific neural progenitor populations. The capability for more targeted irradiation is provided by radiological platforms that combine computer tomography-guided imaging with focal beam irradiation (CFIR) delivery to enable precise anatomical targeting29-36. Radiation beams as small as 0.5 mm in diameter are available to target specific neural progenitor populations35. This methodology allows us to target the hypothalamic ME and arrest proliferation and block neurogenesis in small animals. Following radiological treatment on these progenitor populations, physiological and behavioral tests can be performed to illuminate the potential function of adult-born cells. Focal targeting is especially important for our application since the pituitary gland is located close to the hypothalamic median eminence; irradiation of the pituitary may affect hormonal function and subsequently confound results.

The biological basis for the suppression of neurogenesis following irradiation still remains unclear. Previous radiation studies have relied on large area beams, and have concluded that the suppression of neurogenesis is mediated through an inflammatory response14, 37. As such it is unclear whether highly focal irradiation could suppress neurogenesis, since it does not evoke a substantial inflammatory response. However, recent studies by our group of the classic neurogenic region in the hippocampus have demonstrated that highly focal irradiation with a dose of 10 Gy can suppress neurogenesis for at least 4 weeks after irradiation35.

To interrogate the function of adult-born hypothalamic neurons in the median eminence, we use a precision radiation device capable of delivering CT imaging in combination with small-diameter radiation beams to inhibit ME neurogenesis. Using an X-ray tube attached to a gantry that rotates over a range of 360°, we deliver arc-beam micro irradiation beam with the use of a robotically controlled specimen stage that allows rotation of an animal subject during radiation treatment (Figure 1). A high-resolution X-ray detector is used to acquire images when the gantry is in the horizontal position33. For this study, CT images were reconstructed with an isotropic voxel size of 0.20 mm. On-board CT imaging allowed the identification of a target while the animal is in the treatment position. The target was localized using the CT navigation dose-planning software, which was included with our commercially available radiological platform. After localizing our ROI by CT imaging, the animal was moved to the appropriate treatment position by the robotic specimen stage that has four degrees of freedom (X, Y, Z, θ). Through a combination of gantry and robot stage angles, beams can be delivered from nearly any direction relative to the animal, and stereotactic arc-like treatments are possible29. For these and all other imaging studies, the mice were positioned in an immobilization device that allows delivery of anesthetic isoflurane gas while restricting movement. The immobilization bed is CT compatible, and connects to the robotic specimen stage34.

We expect that CFIR will provide conceptual advances in a number of research areas. Although we use radiological targeting of the hypothalamic median eminence as proof of principle of this technique, CFIR can be used to target any region of the body of any small model organism in principle. In the neurosciences, for example, we envision this technique could be used to evaluate the function of actively proliferative progenitor populations that have been suggested to exist in other circumventricular organs, such as the area postrema38, 39, subfornical organ40, and the pituitary41. Longstanding controversies regarding the functional role of adult neurogenesis and identifying a causal role in behavior can also now be better addressed. In songbird, this technique might address the role of adult neurogenesis in maintaining the robust and seasonal behavior of birdsong42, which has been hampered by the ability to selectively inhibit neurogenesis in specific brain regions. Understanding this robust behavioral model might shed new insight into the role of adult neurogenesis in regulating other sexually dimorphic behaviors. Alternatively, in the metabolic field, CFIR might be used to explore aspects of the role of hepatocyte proliferation and its role in metabolism and energy balance. The possibility for conceptual advance in multiple research disciplines is enhanced by the introduction of this technique.

In this paper, we demonstrate the capabilities of CFIR for precision anatomical targeting of a focal irradiation beam. Although we initially developed this small animal radiation research platform (SARRP) for our studies, other similar devices are now commercially available that can accomplish similar CT-guided focal irradiation43, 44. Hence, we generalize this CFIR protocol with steps required for all research platforms rather than those specific for SARRP. The advantages of CFIR over previous radiological approaches to inhibit neurogenesis are that this technique allows three-dimensional volumetric image guidance for localization and targeting of the dose, conformal dose minimizes exposure to nontargeted brain regions, and high precision beam geometry allows for conformal dose distribution with sharp beam boundaries. We outline how to use CT-guided imaging to target the dose to a specific anatomical region, and upon doing so, how to visualize the radiation dose distribution directly in tissue using immunohistochemical staining for γ-H2AX, a marker of DNA double-stranded breaks35, 45-48. The use of this approach for selective irradiation of neurogenic niches may have significant implications in revealing the functional role of new adult-born neurons on physiology and disease.

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Protocol

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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

Perform film-based calibrations for radiation dose on the radiological platform in advance for each collimator size and type of X-ray tube filter used. In order to target an arc beam of irradiation at the desired target site, use the radiation dose-planning software49 available on most commercial radiation platforms to calculate required delivery time based on the radiation dose, the depth of the region of interest (ROI) based on CT scans of the mouse, and the rotation speed of the robotic platform; details on this are left out of this protocol as they differ between radiation platforms. For the purposes of visualizing the ventrobasal hypothalamus2 (Figure 3) with CT imaging, operate the X-ray tube with a 0.4-mm focal spot and beam energy of 100 kVp with 1 mm Al filtration. For focal irradiation of the HPZ, operate the X-ray tube at 225 kVp, 13 mA, and a 1-mm focal spot with filtration of 0.15 mm Cu, to deliver 10 Gy of radiation at a tissue depth of 0.6 cm over a treatment time of 4.6 min. Targeting between different ROIs will require calculating different parameters.

  1. If studying the effects of high-fat diet on neurogenesis in the median eminence as previously described2, obtain four-week-old female C57BL6/J mice from Jackson Mouse laboratories, and house four mice per cage. Switch food from normal chow to a high-fat diet at five weeks old. Allow mice to acclimate to new housing and food. Perform CFIR treatment at 5.5 weeks old. Transport subjects to the operation room that contains the radiological platform. Minimize stress levels during transfer. Prepare isoflurane gas anesthesia chamber. Then, add a single mouse into chamber. In parallel, prepare heating pad (low setting) for post-treatment.
  2. Once mouse is under and no longer responding to foot-pad compression, bring the subject to the radiological platform and place on the immobilization bed of the robotic stage. Place subject's mouth into the nose-cone anesthesia cup, and teeth into the bite guard (Figure 1D). Lay mouse flat on the immobilization bed and check to see if it continues to maintain unresponsiveness. If so, tape down mouse to the bed with gauze tape. While taping down the mouse to the bed, make sure the head is leveled to a horizontal plane. This can be determined by pulling up the ears and seeing if they are leveled. Once the mouse is in the correct position, close the lead protective shield.
  3. Acquire the computer-tomography scan using on-board software of the experimenter's radiological platform (this will differ between platforms), which will provide a three-dimensional anatomical structural scan of the mouse subject. Check to see if the mouse head is leveled to the horizontal plane. If not, repeat steps 1.2 - 1.3 until the subject's head is leveled.
  4. Identify ROI by CT image. Calculate distance from ROI to the surface of the skull using a 45° angle to the horizontal plane as shown in Figure 3E.
  5. Using on-board software, take an X-ray of the mouse subject from above as shown in Figure 4A. Then, remove mouse from the radiological platform, put on heating pad, and monitor until active.
  6. From the coronal CT images, calculate the average ROI anatomical depth of at least three mice in order to determine delivery dosing. As an example from a previous study2 where 10 Gy of irradiation was administered to the ventrobasal hypothalamus, the depth of the ROI from the skull (from a 45° angle) is 0.66 cm (See Figure 3E). Knowing that, the researchers used the dose planning software (DPS) installed on their radiological platform to calculate the appropriate rotation speed and length of treatment to achieve desired dosage to the ROI.
  7. After determining treatment duration and rotation speed of the robotic stage with the dose-planning software, measure dose distributions of the calculated parameters with GAFchromic radiation-sensitive films embedded in a water-equivalent plastic mock-mouse model. To do this, embed three GAFchromic radiation-sensitive films between four vertically stacked water-equivalent plastic blocks29 as shown in Figure 4B.
  8. Place mock mouse model containing GAFchromic films on robotic stage, and run the focal irradiation beam with the newly calculated parameters. For example, to target the ventrobasal hypothalamus, input the parameters of 0.15 mm Cu filter, 225 kVp, 13 mA, 1-mm diameter radiation beam setting, 45° gantry angle, 1.3°/sec rotation, and 4.6 min to achieve an ROI radiological dose of 10 Gy.
  9. Following irradiation, check films for pattern and intensity of radiation dosage. For a 360° angle rotation with the parameters listed to target the ventrobasal hypothalamus, a dark ring in the film above the isocenter, a small crisp spot at the isocenter film, and a lighter ring in the film below the isocenter corresponding to the cone-beam administration of the irradiation will be observed (Figure 4B).
  10. Superimpose isocenter GAFchromic film over the X-rays of mouse subjects from which the parameters were calculated. The irradiated focal point at isocenter should overlap with the desired ROI region as shown in Figure 4C.

Alternative method: If difficulty in targeting brain ROI persists, use iodine contrast injected intrathecally to enhance visualization of ventricles under CT images. For the sake of brevity, this procedure is left out of this protocol, but is previously described35, 50. Iodine contrast will provide additional ventricular landmarks (Figure 5A).

2. Determining Accuracy of Irradiation Beam

Further confirm CFIR beam accuracy by direct visualization of the radiation beam in tissue2, 35, 51. To do this, perform immunohistochemistry to detect γH2AX 51, a histone protein and an early marker of DNA double strand breaks. Mouse subjects must be transcardially perfused and fixed within one hour of irradiation. Following irradiation, DNA repair rapidly ensues, and levels of ϒH2Ax decrease significantly35.

  1. Repeat steps 1.1 - 1.3.
  2. After the target is identified on CT, the mouse subject is moved under robotic control to align this target with the radiation delivery beam. Input calculated parameters (rotation speed and length of treatment to achieve desired dosage) from step 1.6 into dose-planning software and begin treatment. Treatment is delivered with the gantry pointed to 45° from the vertical while the mouse rotates about a vertically-oriented axis.
  3. Following irradiation, perform transcardial perfusion on mouse subjects. Perform perfusion within one hour52. Following perfusion, immersion fix brains in 4% PFA/PBS and gently rock overnight at 4 °C.
  4. The following morning, wash 5 min in PBS 3x to remove paraformaldehyde fixative. Then, immerse in 30% sucrose in 1x PBS on a rocker at 4 °C.
  5. Gently rock at 4 °C (usually 12-16 hr), until brain sinks to bottom of tube. This serves as a cryoprotection step. Once brains sink, remove brain with a perforated spoon to prevent excess 30% sucrose/1x PBS transfer. Quickly embed in freezing medium on dry ice. Swirl freezing medium with pipette tip and align brain in plastic mold.
  6. Once completely frozen, transfer brain blocks to -20 °C freezer for storage.
  7. On the day when immunohistochemistry will be performed, section coronally at 40 μm thickness, and float into a 24-well plate containing PBS with a thin paintbrush. Sections should be kept in correct serial order for immunostaining to determine irradiation coverage of target region.
  8. Prewarm 0.01 M sodium citrate solution to 80 °C in water bath in preparation for antigen retrieval step. Simultaneously, while the sodium citrate is warming, perform 5 min washes 3x in 0.01 M PBS.
  9. Once sodium citrate solution is at 80 °C, submerge brain sections in the sodium citrate buffer solution. Leave in water bath at 80 °C for 1 hr.
  10. Remove section and allow sodium citrate solution to reach room temperature, then perform three 5-minute washes in 0.01 M PBS.
  11. Block brain sections for 1 hr in PBS-Triton containing 5% normal goat serum.
  12. Incubate brain sections overnight in PBS-Triton containing 5% normal goat serum with 1:700 concentration of mouse anti-phospho-H2AX primary antibody at 4 °C.
  13. The next day, perform 15 min washes 3x in 0.01 M PBS-Triton.
  14. Incubate brain sections PBS-Triton containing 5% normal goat serum with goat anti-mouse secondary antibody conjugated with 488 nm fluorphore at 1:500 concentration for 2 hr.
  15. Perform 15 min washes 3x in 0.01 M PBS-Triton.
  16. Perform 4',6-diamidino-2-phenylindole (DAPI) stain (1:5,000 in PBS) for 10 min to visualize nucleus. Then, wash brain sections for 5 min with PBS.
  17. Mount on electrostatically charged microscope slides by floating section in PBS. Wipe off excess PBS and allow slides to dry. Coverslip the slides using mounting medium and allow the slides to dry in the dark at room temperature overnight.
  18. Take pictures of serial coronal sections with fluorescent microscope. ϒH2Ax immunostaining (green) indicates site of irradiation. DAPI (blue) is a nuclear stain (Figure 5B).

3. Preparation of Mouse Subjects for Focal Irradiation

Examine ϒH2Ax immunostaining results. Once satisfied with the calibration and targeting of the irradiation beam, proceed with the experiment. At this point, the total time required to treat a mouse (from animal setup to completion of beam delivery) is approximately 10-15 min for a 10-Gy treatment with a 1-mm beam.

  1. Order four-week-old C57BL6/J female mice from Jackson Mouse laboratories. House four mice per cage, and switch food from normal chow to a high-fat diet at five weeks old. Ear punch mice to give them unique identifying marks. Monitor health of mice daily. Note: metallic markers cannot be used as they will result in streaking artifacts on the CT.
  2. Weigh mice the day before radiation or sham treatment. Split mice into two cohorts for radiation or sham treatment and ensure that there is no significant difference in weight between cohorts. On the day of treatment, when mice are six weeks old, reweigh all mice and record their mass. Gently transport both cohorts to the radiological platform. Take care to minimize stress levels.
  3. Prepare isoflurane gas anesthesia chamber. Anesthetize two mice, one in the predetermined irradiation group, and another in the sham control group. Prepare heating pad set in the low setting for postoperative treatment.
  4. Follow steps 1.2 - 1.4 for the mouse to receive irradiation. For the sham mouse, keep mouse in anesthesia chamber while treatment is going on. Make sure that anesthesia chamber is near the CFIR platform so any effects on ambient radiation are factored in. After the target is identified on CT, move mouse subject under robotic control to align this target with the radiation delivery beam. Input calculated parameters (rotation speed and length of treatment) into dose-planning software.
  5. Once irradiation treatment is complete, return both sham and irradiated mice to heating pad, and monitor until they wake up.
  6. Return both sham and irradiated mice to animal facility. Monitor every day. Weigh the mice every half week. To confirm irradiation of targeted hypothalamic proliferative zone, administer intraperitoneal injections of BrdU (50 mg/kg) three days post-treatment and examine neurogenesis between groups by colabeling of immunohistochemistry for BrdU and a neuronal marker one month following initial BrdU exposure (Figure 6)2.

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Results

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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...

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Discussion

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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...

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Disclosures

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J.W. has a research funding and consultation agreements with Xstrahl, Inc.

Acknowledgements

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We thank C. Montojo, J. Reyes, and M. Armour for technical advice and assistance. This work was supported by US National Institutes of Health grant F31 NS063550 (to D.A.L.), a Basil O'Connor Starter Scholar Award and grants from the Klingenstein Fund and NARSAD (to S.B.). S.B. is a W.M. Keck Distinguished Young Scholar in Medical Research.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
SARRP research platformXstrahlRS225Ahttp://www.xstrahl.com/xstrahlrs225.htm
SARRP irradiation bunkerXstrahlOptional, but radiation exposure should be contained with alternative lead shielding
GAF chromic filmIPSGAFchromic ETB2
Mouse phantomGammex457Purchase 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 antibodyMillipore, Inc.05-636clone JBW301
High-fat rodent dietResearch DietsD12492i60% of the calories as fat, food should be irradiated
IsofluraneBaxter Healthcare Corporation10019-360-40
0.01 M Sodium citrateFisher Scientific1.471 g of sodium citrate dissolved in 500 ml deionized water
Superfrost Plus slidesFisher Scientific12-550-15
DAPIFisher Scientificnuclear counterstain
Mounting mediumFisher ScientificVectashield or Gelvatol is preferred

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