Manganese-Enhanced Magnetic Resonance Imaging (MEMRI)

Abstract

The use of manganese ions (Mn²⁺) as an MRI contrast agent was introduced over 20 years ago in studies of Mn²⁺ toxicity in anesthetized rats (1). Manganese-enhanced MRI (MEMRI) evolved in the late nineties when Koretsky and associates pioneered the use of MEMRI for brain activity measurements (2) as well as neuronal tract tracing (3). Currently, MEMRI has three primary applications in biological systems: (1) contrast enhancement for anatomical detail, (2) activity-dependent assessment and (3) tracing of neuronal connections or tract tracing. MEMRI relies upon the following three main properties of Mn²⁺: (1) it is a paramagnetic ion that shortens the spin lattice relaxation time constant (T₁) of tissues, where it accumulates and hence functions as an excellent T₁ contrast agent; (2) it is a calcium (Ca²⁺) analog that can enter excitable cells, such as neurons and cardiac cells via voltage-gated Ca²⁺ channels; and (3) once in the cells Mn²⁺ can be transported along axons by microtubule-dependent axonal transport and can also cross synapses trans-synaptically to neighboring neurons. This chapter will emphasize the methodological approaches towards the use of MEMRI in biological systems.

1. Introduction

Mn²⁺ is a trace element essential for normal body function and development throughout the lifespan of mammals (4). Most notably Mn²⁺ is an essential cofactor for several enzymes responsible for a wide variety of physiological body functions (4). Such enzymes include manganese superoxide dismutase (5) which is essential for oxidative stress prevention, pyruvate carboxylase (6) which plays a critical role in gluconeogenesis, arginase (7) which is involved in urea production by the liver, and glutamine synthetase (8), an astrocyte-specific enzyme regulated by about 80% of brain Mn²⁺. The importance of Mn²⁺ is illustrated by studies linking disruption of Mn²⁺ homeostasis to disease occurrence (5, 9). Mn²⁺ deficiency has been linked to deficient bone metabolism in rats (10), as well as skin lesions, bone malformation, epileptic seizures, and increased Ca²⁺ and phosphorous levels in humans (11). Although Mn²⁺ deficiency is clearly associated with adverse effects, the aforementioned studies were achieved with an artificially induced Mn²⁺-deficient diet (11). No naturally occurring Mn²⁺-deficiency related diseases have been observed. However, Mn²⁺ is more frequently of toxicological concern. Although it is considered the least toxic of all essential elements (12), excessive exposure to the metal leads to central nervous system toxicity (4). It has been shown that Mn²⁺ can enter the central nervous system either directly via the olfactory receptor neurons or through the blood brain barrier by diffusion or active transport (13, 14). Once in the nervous system, Mn²⁺ is transported along neurons by microtubule-dependent axonal transport (15, 16) and can traverse synapses to accumulate in neighboring neurons (17, 18). The resulting neurotoxicity preferentially targets the striatum leading to Parkinson’s disease-like symptoms, including generalized bradykinesia, widespread rigidity, tremors, hallucinations, and memory loss (4, 19, 20).

In addition to multiple roles in normal physiology, Mn²⁺ is also a Ca²⁺ analog and can enter excitable cells via several types of Ca²⁺ channels such as voltage-gated Ca²⁺ channels and the Na⁺/Ca²⁺ exchanger (21–24). Mn²⁺ also accumulates in mitochondria via the mitochondrial Ca²⁺ uniporter (25, 26). The analogy of Mn²⁺ with Ca²⁺ resulted in the use of Mn²⁺ as a fura-2 quencher and hence Ca²⁺ indicator in biological systems by fluorescence microscopy (27–31).

Another very important feature of Mn²⁺ is that it is paramagnetic and produces MR contrast by causing a strong reduction in the T₁ relaxation times of water (32–35). Positive contrast is detected in T₁-weighted images of tissues where Mn²⁺ accumulates (32–35).

The combined physical and biological properties of Mn²⁺ make it a useful contrast agent for anatomical and functional imaging in multiple systems. Indeed, manganese-enhanced MRI (MEMRI) has been gaining growing interest in the past few years (2, 3, 36, 37) and currently has three main applications for biological systems. First, owing to its contrast-enhancing properties, systemic Mn²⁺ injections are used for enhancement of the brain cytoarchitecture for anatomical studies (38–44). This technique has been used in adult, as well as in young developing organisms. Its use has further been extended to studying the development of embryos in utero (45). Second, given that Mn²⁺ can enter cells via voltage-gated Ca²⁺ channels, it is used as a marker of activity in specific protocols that promote its accumulation in active brain areas (2, 46–52). This use of MEMRI is termed activation-induced MEMRI or AIM-MRI. AIM-MRI also has applications in the heart because of the high concentration of Ca²⁺ channels (53). This protocol, however, will emphasize MEMRI applications in the nervous system. The third and last application of MEMRI is tract tracing; given that Mn²⁺ is transported by microtubule-dependent axonal transport and can cross synapses to reach post-synaptic neurons, MEMRI has been used as a neuronal tract tracer for several neuronal pathways including the visual, olfactory, and somatosensory pathways, in a variety of animal models, such as mice, rats, monkeys, and birds (3, 54–63). This review will focus on MEMRI applications in rodents. The versatility of MEMRI is also demonstrated by the development of methods for dynamic Mn²⁺ transport imaging, which are proving as useful markers of disease and related therapy (64, 65). The following chapter will expand upon each of the three applications of MEMRI with special emphasis on techniques related to each application.

2. Materials

2.1. Anatomical Contrast Enhancement

2.1.1. Intravenous MnCl₂

1. MnCl₂ as a source of Mn²⁺

2. Sterile water

3. Sterile saline

4. Beaker of warm water for tail warming and dilation of the tail vein

5. 27- or 30-gauge needle

6. Forceps

7. 1-ml syringes

8. Tubing suitable to attach to a 27-gauge needle

9. Pre-anesthetic (e.g., glycopyrrolate)

10. Anesthetic (e.g., isoflurane)

11. Analgesic (e.g., bupivicaine)

12. Sterile saline

13. Tape

14. 4-gauge nylon suture

15. Syringe pump

16. Warming blanket

17. Small animal-monitoring system complete with rectal temperature probe and respiration sensor

18. Neuromuscular blocking agent (pancuronium bromide or gallamine triethiodide)

19. Ventilator

2.2. Activation-Induced MRI

2.2.1. Intravenous MnCl₂

see Section 2.1.1.

2.2.2. Blood Brain Barrier (BBB) Disruption

2.2.2.1. Hyperosmolar Mannitol Infusion

• This requires the infusion of mannitol through the tail vein (or femoral vein). Materials will be identical to Section 2.1 with the exception of using a 5–10% mannitol solution instead of the MnCl₂ solution (66–69).

2.2.2.2. Hyperosmolar Mannitol Injection Through the External Carotid Artery

1. Anesthesia (e.g., isoflurane, urethane or α-chloralose)

2. Tape

3. Hair clipper (Note 1)

4. Surgical tools (blade with holder, hemostat, forceps, scissors etc.)

5. Microvascular clips

6. Disinfecting solutions (e.g., betadine, chlorhexidine, alcohol)

7. Polyethylene tubing PE-50, thinned to an outer diameter of ~0.4 mm (Note 2)

8. PE90 tube attached to the hub of a needle

9. 6-0 nylon suture

10. Mannitol solution (20%)

11. Small metal laryngoscope

12. Muscular blocking agent (pancuronium bromide or gallamine triethiodide)

13. Ventilator

2.2.3. Intraperitoneal MnCl₂ – Visual and Auditory Activation

1. MnCl₂ as a source of Mn²⁺ (66 mg/kg in saline – for intraperitoneal injections) (48, 70).

2. 1-ml syringe.

3. For auditory activation studies: auditory isolation box enabled for auditory stimulation with the addition of a sound synthesizer, audio amplifier, and speakers.

4. For visual activation studies: visual stimulation box consisting of four walls made up of 14-inch computer screens. Remaining areas consist of black-painted wood protected by aluminum mesh.

5. Anesthesia (isoflurane or urethane).

2.2.4. Intranasal MnCl₂ – Olfactory System Activation

1. MnCl₂ as a source of Mn²⁺ (10 mM in H₂O – for intranasal administration of 7 μl/naris or 1.5 M in H₂O for aerosolized Mn²⁺ administration with a vaporizer) (52, 61)

2. Pipette – 10 μl

3. Anesthesia (e.g., isoflurane, urethane)

4. Heating pad

5. Odorant for olfactory stimulation (e.g., 1:100 amyl acetate, 1:10 octanal, 1:10 carvone etc.)

6. Vaporizer

7. Fume hood

2.3. Tract Tracing

2.3.1. Tract Tracing – Visual System

1. MnCl₂ as a source of Mn²⁺ (1 M in H₂O – for intravitreal injections) (36, 62)

2. 27-gauge needle

3. Polyethylene tubing (0.4 mm diameter)

4. A 5-μl Hamilton syringe

5. Anesthesia (isoflurane or ketamine/xylazine combination or pentobarbital sodium, see Note 11)

6. Heating pad

7. Dissecting microscope

2.3.2. Tract Tracing – Olfactory System

1. MnCl₂ as a source of Mn²⁺ (3.79 M in H₂O – for intranasal administration) (61, 65)

2. Pipette – 2 μl or 10 μl

3. Anesthesia (e.g., isoflurane)

4. Heating pad

2.3.3. Tract Tracing – Deep Brain Structures

1. MnCl₂ as a source of Mn²⁺ (5 mM in H₂O – for intracranial injections) (36, 63, 71, 72)

2. Anesthesia (e.g., ketamine/xylazine combination as a pre-operative followed by 2% isoflurane for maintenance)

3. Surgical tools (blade with holder, hemostat, forceps, scissors etc.)

4. Disinfecting solution (chlorhexidine, betadine, alcohol)

5. Small sharp scissors and/or rodent hair clipper

6. Small drill with associated bits (similar to a dental drill)

7. 6-0 nylon suture

8. Mouse/rat brain atlas

9. Capillary tube puller

10. Quartz capillary tubes with filament

11. Surgical area including stereotaxic holder, dissecting microscope and gaseous anesthesia line

12. Surgical tool sterilizer (e.g., glass beads electric sterilizer)

13. Picospritzer with holder and push/pull options (to fill injection needles and subsequently inject solution out of them)

14. Heating pad

15. Sterile cotton swabs

16. Eye ointment

17. Leveling tool (small fork-shaped metallic tool that can be used to ascertain 2D horizontal leveling of the mouse/rat head in the stereotaxic holder)

18. Calibrated volume gauge (Note 3)

3. Methods

3.1. Anatomical Contrast Enhancement

Anatomical contrast enhancement by systemic Mn²⁺ injection has been studied in rodents (38–40, 43, 44), birds (57, 58), and primates (54, 55). The methods presented here are specifically designed for rat brain visualization based upon work developed in Koretsky’s laboratory (44). The following methods can be adapted for use with any organism, provided reasonable optimization is conducted on the organism of interest as well as magnetic field strength.

3.1.1. Preparation of the MnCl₂ Solution

Different concentrations of MnCl₂ can be used in systemic injections for positive contrast in T₁-weighted images (37, 44). Optimization, with regards to the animal model used, as well as available MRI hardware, should be performed for the best results. Also, when preparing MnCl₂, care should be taken as to the tonicity and pH of the final solution. The body fluid has an osmolarity of 300 mOsm/l. One mole of MnCl₂ is equivalent to 3 Osm. Therefore, concentrations in the range of 100 mM should be used to insure proper tonicity when large amounts of MnCl₂ are to be infused to the animals. When adjusting the pH of MnCl₂ solution to a physiological pH of 7.4, bicine buffer, equilibrated with NaOH, is a good buffer. Following are guidelines for the preparation of 100 mM MnCl₂ at pH 7.4, suitable for imaging of rat cytoarchitecture by systemic MnCl₂ injection.

1. Dissolve 1.63 g bicine (FW=163.17) in 100 ml water to obtain a 100 mM bicine solution.

2. Bring solution to pH 7.4 using NaOH.

3. Sterilize solution (either by autoclaving or by filtering).

4. Dissolve 98.95 mg of MnCl₂.4H₂0 (FW=197.91) into 5 ml of sterile bicine solution. Depending on the weight of the animals used, 2–4 ml will be enough for imaging one animal; increase the volume of solution according to the number of animals to be imaged.

3.1.2. Intraperitoneal MnCl₂ Injection

Systemic administration of MnCl₂ by intraperitoneal injection consists of one injection of 100 mM MnCl₂ at a dose of 66 mg/kg. Imaging can be performed as early as 3 h and up to 24 h post-injection.

3.1.3. Setting Up a Tail Vein Line

1. Anesthetize the rat with 4% isoflurane in O₂ initially and then keep it anesthetized with 1.5–2% isoflurane using a facemask.

2. Using forceps, break the metallic part of the 27-gauge needle away from its plastic base connector. Take care to avoid causing the needle to get blocked. Connect the metallic part of the needle to its plastic base using a piece of tubing long enough to allow you to comfortably place the connected syringe onto the pump.

3. Fill 1-ml syringe with sterile physiological saline and attach it to the needle/tubing combination.

4. Immerse the tail in warm water to dilate the tail vein.

5. Insert the tip of the needle into the vein; proper insertion is confirmed by the backflow of blood from the tail into the saline-filled attached tubing.

6. To fix the needle in proper place, first use tape over the tubing to loosely hold everything in place. Then, using the nylon suture, tie a knot around the tail/metallic part of the needle. The suture will not go through the skin.

7. Carefully remove the saline-filled syringe and replace it with a MnCl₂ pre-filled syringe.

8. Place the MnCl₂ syringe into the holder of the syringe pump and set the infusion rate to 1.8 ml/h. Do not start the infusion yet.

3.1.4. MnCl₂ Infusion

1. Inject 0.01 mg/kg glycopyrrolate intramuscularly. Glycopyrrolate is a muscarinic cholinergic blocker used as a pre-anesthetic medication to diminish the risk of vagal inhibition to the heart.

2. Insert a rectal probe into the rat, and maintain the temperature at 37.5°C during the infusion using an animal-heating system (e.g., warming blanket or heated air).

3. Keep the anesthesia light during the infusion (0.5–1% isoflurane).

4. The goal MnCl₂ concentration is 175 mg/kg, which amounts to approximately 2 ml of total volume per animal (Note 4).

5. To avoid dehydration, inject sterile saline subcutaneously (6.7 ml/100 g) immediately and 6 h after the MnCl₂ infusion.

6. Keep the animals under controlled temperature for up to 24 h post-infusion. It is normal for the animals to display lethargic behavior at the end of the MnCl₂ infusion. Their behavior will gradually improve to normal by 24 h post-infusion.

3.1.5. Animal Preparation for MRI

1. Anesthetize rats with 4% isoflurane initially.

2. Intubate the animals and keep them ventilated with 1.5% isoflurane in O₂ (see Section 3.2.1.1. for detailed intubation protocol).

3. Maintain body temperature at 37.5°C using an animal-heating system.

4. Monitor temperature, blood pressure, and respiration rate with a small animal physiological monitoring system.

5. Inject the animals with pancuronium bromide (2.5 mg/kg) intraperitoneally to suppress motion (an alternative neuromuscular blocking agent is gallamine triethiodide 80 mg/kg i.v.).

3.1.6. Imaging Parameters

It should be noted that imaging protocols and parameters will vary considerably depending on the field strength to be used. Reported below are the optimal imaging parameters for proton MRI on an 11.7 T magnet based upon the work done by Aoki et al (44). The following parameters can be used as a starting point; however, further optimization should be performed for different field strengths and imaging protocols.

• Two-dimensional multi-slice multi-echo (MSME-2D)

• Repetition time (TR) = 300 ms

• Echo time (TE) = 10.5 ms

• Matrix size = 256 × 256

• Field of view (FOV) = 25.6 × 25.6 mm

• Slice thickness (ST) = 1 mm

• Number of averages (NEX) = 8

An inversion recovery sequence could be used to acquire T₁-weighted images as well. The parameters are as follows:

• Inversion time = 1,100 ms

• TR = 4,000 ms

• TE = 11.2 ms

• Matrix size = 512 × 256

• FOV = 38.4 × 19.2 mm

• ST = 1 mm

• NEX = 1

• Three-dimensional spin echo (SE-3D)

• TR = 250 ms

• TE = 7.3 ms

• Matrix size = 256 × 256 × 128

• FOV = 19.2 × 19.2 × 9.6 mm

• NEX = 2

• Total acquisition time = 273 min

3.1.7. Expected Results

The expected results are an increase in positive contrast enhancement in the central nervous system (Fig. 7.1). The pattern of enhancement can be obtained over a wide range of MnCl₂ concentrations, with regions of the brain lacking a blood brain barrier (BBB), such as the pituitary gland, exhibiting stronger enhancement at low doses. Regions with an intact BBB, such as the hippocampus and cortex show a dose-dependent increase in contrast enhancement. Higher doses may even allow the detection of finer details of the neuroarchitecture, such as cortical laminae structure.

Fig. 7.1.

T₁-weighted MRI after systemic MnCl₂ administration in the rat. T₁-weighted MRI of a control rat (column A) and a rat 1 day after IV infusion of MnCl₂ solution (column B). Top row shows transverse slices at the level of the olfactory bulb (OB, Bregma: +7 mm). The middle row shows horizontal slices including the hippocampal formation (Bregma: −6 mm). The bottom row shows sagittal slices. The signal intensity of the T₁-weighted MRI was enhanced prominently 1 day after systemic administration of MnCl₂ in the rat. There were characteristic signal enhancements that were large in the olfactory bulb (OB), hippocampus, cerebellum, and pituitary. Reprinted from Aoki et al. (44), copyright 2004, with permission from Elsevier.

3.2. AIM-MRI

Activation-induced MRI (AIM) is essentially a Mn²⁺-based functional MRI paradigm. It was introduced in 1997 by Lin and Koretsky as a blood-flow independent alternative to functional MARI (fMRI) (2). This method is based on the following two essential properties of Mn²⁺: (1) Mn²⁺ can enter the brain parenchyma from the blood via a disrupted (or leaky) blood brain barrier (BBB) (66) and (2) Mn²⁺ is a Ca²⁺ analog that can enter neurons via voltage-gated Ca²⁺ channels and accumulates in neurons in an activity-dependent manner (30, 73–75). Mn²⁺ ions cannot efficiently enter the brain parenchyma through an intact BBB (66). Some diffusion may occur at the blood/CSF interface in choroid plexuses, but the amount of Mn²⁺ entering the brain is minimal compared to the cases where the BBB is disrupted (1, 76, 77). As a result, most AIM studies to date were performed in conjunction with BBB disruption (66). Some studies on the activation of the auditory (48, 78, 79) and visual pathways (70, 80) following auditory and visual stimulation respectively were performed in mice without disruption of the BBB. Also, a subset of functional studies in the olfactory system, capitalized on the active entry of Mn²⁺ through Ca²⁺ channels and its trans-synaptic transport (61). These studies were a spin-off from tract-tracing studies and were also performed without BBB disruption.

Several groups have used this technique since 1997 with certain variations regarding the route of administration of MnCl₂ as well as the paradigm followed to break the BBB. Protocols are presented here for intravenous or intraperitoneal MnCl₂ administration with hyperosmolar mannitol injections and infusions to break the BBB. Protocols involving olfactory, visual, or auditory activation without BBB disruption are also presented.

3.2.1. Intravenous Infusion of MnCl₂ with BBB Disruption

The protocol for preparing MnCl₂ and setting up a tail vein line for intravenous MnCl₂ infusion was described in Sections 3.1.1–3.1.3. Here, a protocol for disrupting the BBB in conjunction with MnCl₂ infusion is described.

3.2.1.1. Direct Oral/Tracheal Intubation for Artificial Ventilation (Note 5)

1. Anesthetize the mouse/rat with an intraperitoneal injection of α-chloralose and urethane combination (Note 6).

2. Upon lack of toe pinch reflex, place the animal in dorsal position on a pre-heated warming pad.

3. Tape the limbs down.

4. Pull the head back by placing 4-0 nylon behind the upper incisors.

5. Use a small metal laryngoscope to pull the lower jaw down and expose the tracheal opening.

6. Insert a ~2-cm long PE-90 tubing attached to the hub of a needle about 3 mm into the trachea. The tip of the tube should be beveled in the direction of the natural bend of the tubing to avoid any tissue damage during insertion (Note 7).

7. Attach the needle hub to a ventilator and set it to 80–100 breaths/min. (Do not turn the ventilator on yet; this will occur when imaging begins.)

3.2.1.2. Catheterization of the External Carotid Artery for Hyperosmolar Mannitol Injection

1. While the animal is still in dorsal position from the previous step, shave the neck area with a hair clipper (for rats) or sharp small scissors (for mice) (Note 1).

2. Disinfect the operating field with betadine and 75% alcohol.

3. Make a vertical 5-cm incision and expose the arteries.

4. Temporarily clamp the right common and internal carotids using the microvascular clamps.

5. Carefully insert the PE50 tubing into the external carotid artery through a small puncture in the retrograde direction (Note 8).

6. Secure the tubing in place with 6-0 nylon suture. Tighten the sutures around the tube very well to prevent any further bleeding from the artery.

7. Remove the microvascular clamps.

8. Sew the wound back roughly keeping the tubes well in place.

9. The mouse is now ready to be placed in the magnet.

3.2.1.3. Animal Preparation for MRI

1. Place the animal in a suitable holder with bite bars.

2. Set up a tail vein line for MnCl₂ infusion as described in Section 3.1.2 (Note 9).

3. Extend all tubing outside of the magnet room.

4. Maintain anesthesia with 2% isoflurane.

5. Inject the animal with pancuronium bromide (2.5 mg/kg) intraperitoneally to suppress motion (an alternative neuromuscular blocking agent is gallamine triethiodide 80 mg/kg i.v.).

6. Start the ventilator for artificial breathing (80–100 breaths/min).

3.2.1.4. Experimental Protocol

Keeping in mind that there are several different variations of experimental protocols, a typical setting for AIM experiments is given below.

1. Acquire a series of baseline scans.

2. Start the MnCl₂ infusion (typically infusions last about 1 h). Care should be taken in selecting the concentration of MnCl₂ for AIM studies. MnCl₂ causes toxicity to the heart due to Mn²⁺ homology to Ca²⁺. A very common initial effect of MnCl₂ is a drop in blood pressure that is typically recovered to normal within 10 min. Therefore, the concentration of MnCl₂ may not have drastic effects in long-term studies, but it does affect short-term studies such as AIM. A concentration of 0.2 mmol/kg infused over an hour has been shown to work well.

3. About halfway through the infusion, inject a bolus of 25% mannitol (5–7 ml/kg) through the external carotid catheter. Keep the room temperature higher than 25°C and pre-warm all tubing and attachments related to the mannitol injection. This is necessary to prevent mannitol recrystallization and subsequent micro-infarcts (Note 10). Unilateral injection usually results in the opening of the BBB on the side of the injection, with the contralateral receiving less Mn²⁺. The inhomogeneous opening of the BBB is not very well understood, but modulating some factors such as dose of mannitol, injection rate, distance of the injection site from the artery, age of the animal etc. plays an important role in obtaining sufficient BBB disruption for suitable contrast achievement.

4. At the completion of the MnCl₂ infusion, administer the activating signal specific for the AIM study at hand (pharmacological as well as behavioral stimuli have been described).

5. Begin T₁-weighted image acquisitions at different time intervals to measure activation of specific brain areas involved in your study.

3.2.1.5. Imaging Parameters

Different imaging paradigms are possible for nervous system activity measurements using MEMRI in conjunction with BBB disruption. The imaging parameters will depend on the area of interest, organism used, as well as magnetic field strength. Optimization with regards to all of these variables is required for best results. Following is an example of imaging parameters, excerpted from Weng et al (50), for imaging cortical activity in rats following whisker stimulation at 3T.

• Multi-slice spin-echo sequence

• TR = 500 ms

• TE = 10 ms

• In-plane resolution = 187 μm

• ST = 1.5 mm

3.2.1.6. Expected Results

Depending upon the system under study, one can expect to detect increased signal enhancement in areas of the brain involved in the activity studied. For example, following whisker stimulation, Weng et al. observed signal enhancement in the right cortical barrels of the rat brain (Fig. 7.2) (50).

Fig. 7.2.

Three consecutive slices of the averaged Mn²⁺-enhanced T₁WIs under urethane anesthesia a. Mn²⁺ enhancement was observed in right cortical barrels. The color maps of the averaged Mn²⁺-enhanced T₁WIs b. Reprinted from Weng et al. (50), copyright 2007, with permission from Elsevier.

3.2.2. Activation Studies Without BBB Disruption – Visual and Auditory Systems

3.2.2.1. Activation Studies in the Auditory System

3.2.2.1.1. MnCl₂ Administration

1. Administer 66 mg/kg MnCl₂ as an intraperitoneal injection.

2. Place the animals in normal isolation cages with free access to food and water.

3. Place the isolation cage (with the animal in it) inside an auditory isolation box.

4. Subject the animals to different sound stimulation protocols over a period of 24 h.

5. Upon completion of the auditory stimulation paradigm, initially anesthetize the animals with 5% isoflurane followed by maintenance with 2% isoflurane and acquire T₁-weighted images.

6. A target region of interest for MEMRI quantification is in the auditory nuclei.

3.2.2.1.2. Imaging Parameters

Different imaging paradigms are possible for activity measurements from the auditory system using MEMRI. Following are optimal imaging parameters for the acquisition of T₁-weighted images of the mouse brain at 7T. These parameters are adapted from the work of Daniel Turnbull and associates (48) studying nerve activity in the auditory pathway of mice and are meant to be a starting guide. Further optimization is required depending upon the animal model used as well as the magnetic field strength.

• 3D gradient echo pulse sequence

• TR = 50 ms

• TE = 4 ms

• Flip angle = 65°

• Total imaging time = 1 h 50 min per mouse

This sequence yields a volumetric image set covering the whole brain, with an isotropic resolution of 100 μm

3.2.2.1.3. Expected Results

With the described auditory stimulation protocol combined with a systemic MnCl₂ administration, one can expect to observe significant Mn²⁺ enhancement in structures of the auditory system, including the auditory nuclei in the brainstem and the thalamus (Fig. 7.3). Although the auditory cortex would also be expected to show enhancement, this particular protocol does not lead to signal enhancement in the auditory cortex, perhaps due to less Mn²⁺ reaching its remote location (48). Nonetheless, this technique is a very useful approach for studying neural activation in the auditory system and can be applied to different animal models of diseases involving the auditory system (e.g. Fig. 7.3).

Fig. 7.3.

MEMRI enhancement in brainstem auditory nuclei was altered in mice with conductive hearing loss (CHL). Comparisons of individual mice with bilateral CHL (bi-CHL) (a), mice with unilateral CHL (uni-CHL) (b), and normal mice (c) demonstrated marked differences in MEMRI signals in the cochlear nucleus (CN) (arrow heads) and inferior colliculus (IC) (arrows), but not in non-auditory caudate putamen (CPu). Adapted by permission from (48), copyright 2005.

3.2.2.2. Activation Studies in the Visual System

3.2.2.2.1. MnCl₂ Administration

1. Prior to the start of the procedure, house the animals in darkness for 8–12 h. All subsequent procedures and animal handling should be performed in darkness or under very dim red light.

2. Administer 66 mg/kg MnCl₂ as an intraperitoneal injection.

3. Place the animal in the stimulation chamber for 8 h. Stimulation consists of a moving square wave (as opposed to constant diffuse light) to avoid habituation.

4. Upon completion of the stimulation paradigm, anesthetize the animals with isoflurane or urethane and acquire T₁-weighted MR images.

3.2.2.2.2. Imaging Parameters

Different imaging paradigms are possible for activity measurements from the visual system using MEMRI. Following are two sets of optimal imaging parameters for the acquisition of T₁-weighted images of the mouse or rat brain at 4.7T. These parameters are adapted from the work of Bruce Berkowitz and associates (70, 80) studying visual system activity in awake animals and are meant to be a starting guide. Further optimization is required depending on the animal model used as well as the magnetic field strength.

3.2.2.2.3. Mouse Brain

• Adiabatic spin echo sequence

• TR = 350 s

• TE = 16.7 ms

• Number of acquisitions = 16

• Sweep width = 61,728 Hz

• Matrix size = 512 × 512

• ST = 620 μm

• FOV = 12 × 12 mm²

3.2.2.2.4. Rat Brain

• Rapid Acquisition with Relaxation Enhancement (RARE) sequence

• TR = 330 ms

• TE = 16.6 ms

• RARE factor = 8

• Number of acquisitions = 2

• Matrix size = 256 × 256 × 173

• FOV = 3.84 × 3.84 cm²

• ST = 150 μm

• Time = 80 min/image

3.2.2.2.5. Expected Results

With the aforementioned protocol, one can expect to detect Mn²⁺ enhancement consistent with layer-specific visual cortex activity in awake and free-moving animals. Layers of a given cortical region respond differently to sensory stimulation and this MEMRI protocol appears to be sensitive enough to detect subtle changes in layer-specific activity (70, 80).

3.2.3. Activation Studies Without BBB Disruption – Olfactory System

3.2.3.1. MnCl₂ Administration

Two current Mn²⁺ exposure modalities exist for activation studies from the olfactory system:

• Intranasal administration of MnCl₂

• 1Anesthetize the animal with 5% isoflurane.
• 2Pipet 7 μl of a 10 mM MnCl₂ solution in each naris.
• 3Allow the animal to recover on a heating pad.
• 4Place the animal in a clean cage and drop 7 μl of odorant solution in each of the four corners of the cage.
• 5Allow odorant exposure for 20 min.
• 6Anesthetize the animal with 5% isoflurane for imaging. Exposure to aerosolized MnCl₂
• 1Prepare a 1.5 M solution of MnCl₂ in H₂O.
• 2Place the solution in the heating chamber of a humidifier or vaporizer. For experiments involving the exposure to an odor, mix the odor within the MnCl₂ solution. Use a different humidifier for every odor used to avoid cross-contamination between experiments.
• 3Place the vaporizer inside a fume hood to avoid exposure to Mn²⁺ vapors.
• 4Animal exposure to the aerosolized MnCl₂ with or without odor is performed on either awake or anesthetized animals and is done as follows:

  • For awake animals: place the animal in the same plastic box housing the humidifier in the hood. Turn the humidifier on for 30 min. Keep the mouse in the box for 1.5 h after the humidifier has been turned off. It is important not to open the box during that time because of possible exposure to the aerosolized Mn²⁺ still present.

  • For anesthetized animals: anesthetize the animal with 20 mg/kg urethane and secure it on top of the humidifier with a restraining device. The exposure paradigm involves two sequences of 5-min on and 5-min off, and then the animal is kept for 1.5 h in the chamber with the humidifier off. Again, it is important not to open the box during that time because of possible exposure to the aerosolized Mn²⁺ still present.

3.2.3.2. Imaging Parameters

Different field strengths will dictate different imaging parameters for best results. The following parameters are adapted from the work of Alan Koretsky and associates (52) for studying olfactory activation in mice at 11.7T. These imaging parameters are meant to be a starting guide; further optimization with regards to field strength and organism used is required.

• T₁-weighted images acquired by a 3D RARE sequence

• TR = 300 ms

• TE = 10 ms

• Matrix size = 128 × 128 × 64

• RARE factor = 2

• Isotropic spatial resolution = 100 μm

3.2.3.3. Expected Results

The expected results from the activity-dependent olfactory tract tracing are a gradual increase in signal enhancement ranging from the olfactory epithelium to the olfactory bulbs. Signal enhancement will follow a region-specific enhancement depending on the stimulating odorant used (Fig. 7.4).

Fig. 7.4.

Detecting odor-dependent Mn²⁺ enhancement in mouse olfactory bulb by MRI. MEMRI maps after stimulation by acetophenone, carvone, octanal, and control in four mice, respectively, show distributed enhancement in the glomerular layer with each odorant having its own distinct spatial pattern. High signal change at the interface between the olfactory nerve layer and olfactory turbinates (arrow) indicates where Mn²⁺ flowed in. Scale bars represent 1 mm. Reprinted from Chuang et al. (52), copyright 2009, with permission from Elsevier.

3.3. Tract Tracing

Tract tracing takes advantage of the following two properties of Mn²⁺: (1) it is transported along neurons by microtubule-dependent axonal transport and (2) it can traverse synapses and reach second-order neurons leading to contrast enhancement of the whole neuronal system in question. Tract-tracing studies have been performed in several systems such as the visual and olfactory systems as well as from deep brain structures such as the hippocampus and amygdala.

3.3.1. Tracing the Visual Pathway

3.3.1.1. MnCl₂ Administration

1. Anesthetize the animal initially with 5% isoflurane and then maintain it with 2% isoflurane.

2. Place the animal in the prone position on a heating pad to maintain body temperature

3. Gently detach the metallic piece of the 27-gauge needle from its plastic hub using forceps. Connect the metallic portion of the needle to the Hamilton syringe via a small piece of polyethylene tubing.

4. Insert the tip of the needle into the vitreous with the aid of a microscope. A good injection site is about 2 mm posterior to the dorsal limbus.

5. Inject 0.1 μl of the MnCl₂ solution over 5 min. The volume injected can be gauged by the advancement of the meniscus in the polyethylene tube using the scale of the Hamilton syringe.

6. Leave the injection needle in the eye for at least 15 min and then withdraw it very slowly to minimize the loss of MnCl₂ through leakage from the injection site. This waiting time is necessary to insure homogenous distribution of the MnCl₂ inside the eye as well as for the intraocular pressure to reach equilibrium.

7. Terminate anesthesia and return the animals to their cages.

8. Following the MnCl₂ injection and prior to imaging, it is advised to check the integrity of the eyes. Successful injection can be ascertained by a bright-looking vitreal humor on a T₁-weighted image.

3.3.1.2. Imaging Parameters

Imaging parameters will vary depending on the animal model used as well as on the magnetic field strength. Following are the optimal imaging parameters used by Watanabe et al. to trace the visual pathway of rats at 2.35T (62). These parameters should be used as a starting guide and further optimization with regards to field strength performed for best results.

• T₁-weighted 3D FLASH gradient echo sequence

• TR = 15 ms

• TE = 4.2 ms

• Flip angle = 25°

• FOV = 50 × 50 × 16 mm

• Matrix size = 256 × 256 × 128

• NEX = 8

• Acquisition time = 65.5 min.

3.3.1.3. Expected Results

Signal enhancement is expected to be seen in the entire visual pathway, starting from the eye and extending to the superior colliculus. An example of such enhancement is illustrated in Fig. 7.5 (62).

Fig. 7.5.

Signal enhancement of the rat visual pathway (24 h after Mn²⁺-injection into the left eye) in oblique sections 235° (top left), 210° (top right), 15° (bottom left), and 137.5° (bottom right) relative to a transverse reference plane. Enhanced structures are (1) left retina, (2) left optic nerve, (3) optic chiasm, (4) right optic tract, (5) right lateral geniculate nucleus, (6) right brachium of the superior colliculus, (7) right pretectal region, and (8) right superior colliculus. Reprinted from (62), copyright 2001, with permission from John Wiley & Sons, Inc.

3.3.2. Tracing the Olfactory Pathway

3.3.2.1. MnCl₂ Administration

1. Anesthetize the animal with 5% isoflurane.

2. Hold animal in a vertical position by slightly pinching the hair in the back of the head.

3. Using a 10-μl pipette, administer 2 μl of a 3.9 M MnCl₂ solution to each naris. The 2 μl can be either administered as 2 × 1 μl or at once. It is normal to observe some bubbling from the nose following the nasal lavage.

4. Place the animal on a heating pad to accelerate recovery. Usually it only takes a few minutes for the animal to regain consciousness.

5. Return the animal to the housing cage.

6. Proper lavage can be ascertained by very dark-looking turbinates on a T₁-weighted image (due to T₂ effects of the concentrated Mn²⁺ solution).

3.3.2.2. Imaging Parameters

Based upon the work of Pautler et al (3), the optimal imaging parameters for tracing the olfactory system of the mouse at 7T are as follows:

• T₁-weighted multi-slice spin echo sequence

• TR = 307 ms

• TE = 12.7 ms

• FOV = 2.5 cm

• ST = 1 mm

• Matrix size = 128 × 128

Higher resolution 3D scans can be acquired using the following parameters:

• TR = 300 ms

• TE = 8.7 ms

• FOV = 2.5 × 2.5 × 2.5 cm

• Matrix size = 128 × 128 × 128

3.3.2.3. Expected Results

The expected results are a positive contrast enhancement in the olfactory bulbs as well as the primary olfactory cortex as illustrated in Fig. 7.6 (3).

Fig. 7.6.

a Three sagittal slices of a mouse treated with Mn²⁺ in the naris from a representative 3D T₁-weighted MRI sequence. Note the highlighting of the olfactory bulb as well as the primary olfactory cortex leading from the bulbs. b Four axial slices from the same mouse treated with Mn²⁺ in the naris from a 3D T₁-weighted MRI sequence. Note the highlighting of the outer layers of the olfactory bulbs where the olfactory glomeruli are located. In addition, the enhanced contrast continues caudally into the primary olfactory cortex. Due to the length of the scan, mice were sacrificed before 3D imaging. Reprinted from (3), copyright 1998, with permission from John Wiley & Sons, Inc.

3.3.3. Tracing of Deep Brain Structures

3.3.3.1. Injection Site Coordinates

1. Identify the brain region you wish to inject.

2. Utilizing a stereotaxic brain atlas, determine the stereotaxic coordinates of the region of interest. This region will most likely encompass multiple slices. Be sure to choose the coordinates that correspond with the largest region in the structure of interest if possible. Additionally, it should be noted that the stereotaxic coordinates will vary based upon sex, age, and animal strain.

3.3.3.2. Preparing the Injection Needle

1. Using a pipet puller and a quartz capillary tube with filament, pull the tube to create the injection needle. A long, fine-tip needle is needed. Micropipette pullers use a size) velocity (determines the point at which the heat is turned off), delay (the time between deactivation of the laser and the application of a hard pull), and pull (final hard pull applied on the capillary tube) to create needles of different shapes and lengths. Keeping in mind that different pullers operate under different settings (same pullers may even differ depending on the type of filament they have), the following parameters are good starting points:

   Heat=700; Filament=3; Velocity=60; Delay=140; Pull=175

2. Using fine forceps, gently break the tip of the needle to open it (the needle comes out sealed from the puller).

3. Place the needle in the picospritzer holder.

4. Set the picospritzer to the “pull” option and slowly fill the needle with the MnCl₂ solution.

5. Set the prepared needle aside and proceed to preparing the animal for surgery.

6. It is advisable to pull and fill several needles, in case one breaks during the surgery. Store the filled needles in a humidified chamber to prevent crystallization of the MnCl₂ solution.

3.3.3.3. Surgery

1. Anesthetize the animal with pentobarbital sodium or ketamine/xylazine combination (Note 11).

2. Upon lack of toe pinch reflex, place the animal on a warming blanket and clip the hair on the back of the head (the area extending from between the ears to the start of the back) (see Note 1).

3. Fix the animal’s head in a stereotaxic holder complete with a bite bar and cheek/ear bars.

4. Maintain anesthesia with 2% isoflurane.

5. Clean the operating field with the disinfecting solution chlorhexidine alternating with sterile water (three times).

6. Make a vertical incision extending from the nose to the start of the back; hold the skin open with hemostats.

7. With the help of the leveling tool and the microscope, make sure that the head is leveled properly both in the longitudinal and horizontal directions.

8. Using the stereotaxic device, determine the coordinates of your animal’s Bregma.

9. Calculate the placement of your region of interest with regards to the Bregma coordinates. For example, if your region of interest mesolateral position was −4.2 and your animal’s Bregma was located at the mesolateral coordinate of 34.6, then you will need to place your holder at the 30.4 position (34.6–4.2). The same type of calculation applies to the other coordinates.

10. Mark the 2D location of the injection site by making a small scrape with the tip of a 27-gauge needle.

11. Begin drilling carefully at that location. Be sure to only drill a hole into the skull (e.g., mouse skull is less than 1 mm thick). The drill bit should not go through the dura or brain tissue.

12. Attach the volume gauge to the pre-filled needle with a small piece of modeling clay.

13. Lower the filled needle into the drilled hole. Carefully let the tip of the needle pierce the dura. Keep lowering until you reach the pre-calculated depth coordinate of your region of interest.

14. Adjust the microscope focus on the gauge so that the injected volume can be monitored.

15. Set the injection pressure to 20 psi approximately and begin with an injection time of 5 ms. Gradually increase the injection time until you see the meniscus slightly move within the needle.

16. Inject the full volume using this setting. Typically 10–20 nl is suitable for tracing from deep brain structures (see Note 3).

17. Leave the injection needle in for at least 5 min and then withdraw it very slowly. This step is necessary to avoid the backflow of MnCl₂ through the injection canal.

18. Place a few drops of analgesic such as bupivicaine just underneath the scalp and away from the drill hole and then suture the wound with 6-0 nylon suture.

3.3.3.4. Imaging Parameters

Different imaging paradigms are possible for tracing from deep brain structures following setereotaxic Mn²⁺ injections. Both 2D and 3D protocols can be used (3D recommended). Following are optimal imaging parameters for the acquisition of T₁-weighted images of the guinea pig brain at 3T. These parameters are adapted from the work of Lee et al. for tracing the auditory pathway in guinea pigs (63) and are meant to be a starting guide. Further optimization is required depending on the organism used as well as on the magnetic field strength.

• Two-dimensional spin echo sequence

• TR = 450 ms

• TE = 13 ms

• Matrix size = 256 × 256

• FOV = 50 × 50 mm

• ST = 1.5 mm (with a slice gap of 0.1 mm)

• NEX = 10

• Three-dimensional gradient echo sequence

• TR = 10.2 ms

• TE = 2.5 ms

• Flip angle = 30°

• Matrix size = 256 × 256 × 128

• FOV = 50 × 50 × 50 mm

• NEX = 7

3.3.3.5. Expected Results

The expected results are a multi-synaptic tract tracing to all structures involved in the system that is peripherally injected. For example, following injection of Mn²⁺ to the cochlea (63), signal enhancement can be observed in the entire auditory pathway, including the cochlear nucleus, the lateral lemniscus, the inferior colliculus, the medial geniculate nucleus, and the trigeminal tract (Fig. 7.7).

Fig. 7.7.

T₁-weighted, 2D spin-echo MR image (A) before MnCl₂ administration and T₁-weighted, 3D gradient-echo image (B) after 12 h of MnCl₂ administration at the left cochlea in the guinea pig. The images’ orientation was obtained at the coronal section, and the voxel resolution was 195×195×200 μm (3). The post-injection image shows signal enhancement of the auditory pathway. Enhanced structures are as follows: (a) cochlear nucleus (CN), (b) lateral lemniscus (LL), (c) inferior colliculus (IC), (d) medial geniculate nucleus (MGN), and (e) trigeminal tract (TT). Reprinted from Lee et al. (63), copyright 2007, with permission from Elsevier.

3.4. Concluding Remarks

As delineated by the multitude of techniques described in this chapter, MEMRI is undoubtedly a very useful technique for the study of the brain anatomy and activity. Perhaps the most important aspect of MEMRI is that it is minimally invasive and offers the possibility of longitudinal studies. This is of utmost importance for efficient diagnosis and understanding of disease states. Although MEMRI has been quite developed and refined in several organ systems (37, 53) of various animal models such as rodents (3, 44, 81, 82), song-birds (56–58), and non-human primates (55, 83), its use in humans is still almost non-existent. To date, Mn²⁺ has been approved for clinical imaging only in its chelated form (84). This is primarily due to the toxicity associated with a high concentration of free Mn²⁺ ions. High concentrations of Mn²⁺ have been shown to cause acute cardiovascular depression (85) as well as neurodegenerative damage to the nervous system (4). Many efforts are currently focused on developing Mn²⁺ contrast agents lacking the traditional side effects of Mn²⁺. One such agent, available from Eagle Vision Pharmaceuticals, consists of free Mn²⁺ ions formulated with Ca²⁺ to override the transient effect of Mn²⁺ as a Ca²⁺ competitive inhibitor. This agent is currently used in dogs and pigs for cardiac (86, 87) as well as for vascular imaging (88). The development of such agent shows promise for the imminent use of Mn²⁺ as a clinical contrast agent for cardiac and brain imaging.