Now, 2008

8.6 Perivenous syndrome

First, all sons and daughters, take your father out to dinner on Sunday.

Happy Father's Day!

We will now touch upon the "perivenous syndrome". Perhaps the best explanation is that in Prof Philip James's " Hyperbaric oxygenation in fluid microembolism", Neurol Res, 29(2): 156-161(6), 2007:

[Abstract] "...Studies of bubbles formed on decompression in diving have demonstrated the importance of pulmonary filtration in the protection of the nervous system and that filtration is size dependant, as small bubbles may escape entrapment. Fluid and even small solid emboli, arresting in or passing through the cerebral circulation, do not cause infarction, but disturb the blood-brain barrier inducing what has been termed the 'perivenous syndrome'. The nutrition of areas of the white matter of both the cerebral medulla and the spinal cord depends on long draining veins which have been shown to have surrounding capillary free zones. Because of the high oxygen extraction in the microcirculation of the gray matter of the central nervous system, the venous blood has low oxygen content. When this is reduced further by embolic events, tissue oxygenation may fall to critically low levels, leading to blood-brain barrier dysfunction, inflammation, demyelination and eventually, axonal damage. These are the hallmarks of the early lesions of multiple sclerosis where MR spectroscopy has also shown the presence of lactic acid..."

Having already gone over the principle and practice of NMR, now a quick review of the current treatment of multiple sclerosis (MS):

MS, despites years of research, still does not have an agreed upon etiology. Since the principal symptom is inflammation, treatments thus far have targeted it by using anti-inflammatories. For acute relapses, patients with relapse-remitting MS are treated with steroids usually intravenous methylprednisolone (IVMP). For longer-term maintenance, there are the interferons, IFN1a and INF1b, that can block immune factors known as class II MHC molecules. With this treatment, the T cells can no longer breach the blood-brain barrier to cause cerebral damages.

The search for a better alternative to interferons culminated in 2004 when natalizumab, a monoclonal antibody, was approved for MS treatment. However, shortly after, reports of a rare progressive multifocal leukoencephalopathy (PML) surfaced. This new drug was withdrawn from the market in Feb, 2005. And after extensive reviews, in June, 2006, the FDA allowed natalizumab to return to the market with certain safety restrictions. The benefits of natalizumab, either by itself or in conjunction with IFNa1 seem to outweigh the risks according to several 2006 reports. So MS patients actually do have treatment choices and many are well-managed by their neurologists.

Of course, there are always other drugs being developed and tested, for example, the immunosuppressants, the statins, estrogen, even the cannabinoids. Also being tested are stem-cell implants and plasmapheresis. Preliminary results are somewhat mixed in most if not all cases.

Most intriguing, however, is the hyperbaric oxygen treatment (HBOT) of MS. In fact, the efficacy of HBOT has been reported in as early as 1983 (for details, see: Fischer BH et al. Hyperbaric oxygen in the treatment of multiple sclerosis; a randomised placebo-controlled double-blind trial. New Eng J Med 1983;308:181-186).

Because of the venous leakage that causes inflammation in the surrounding area, the treatment principle is to provide the repairing process the best environment before sclerosis sets in. And one of the major environmental factors is indeed oxygen. Logically, then HBOT should be employed urgently as it it the best way of delivering oxygen.

Treating early stage MS is now possible with the advent of MRI and MRS (and eventually, the 4-dimensional NMR spectroscopy). To firmly establish HBOT as the treatment of choice, large-scale double-blind studies based on MR technology must be conducted. The focus will be on the level of lactate, the 1.35-ppm resonance [note: its chemical shift is pH-dependent], th index of anaerobic glucose metabolism.

In fact, all eye doctors should be on the lookout for patients with early MS - pay more attention to a complaint of diplopia for one thing, and do take retrobulbar pains seriously. A judicious referral for MRI scan is mandatory. The treatment choices also must be presented and explained to the patients.

Ideally, those microemboli that cause focal ischemia in MS in the first place should be definitely identified and the method for their removal developed. Something for the near future, perhaps.

Lest we forget: many devastating ocular diseases are ischemic in nature, for example, primary open-angle glaucoma, age-related macular degeneration, and diabetic retinopathy. Is there a role for HBOT in the treatment of these diseases? You tell me.

8.5 Optic neuritis

(The above is a fundus photo of optic neuritis, notice the blurred disc margin. This is not always seen, however. Image from meded.ucsd.edu.)

Sometimes a patient's chief complaint is this: Over the past few weeks, vision fades in and out and the patient is now experiencing "pain behind the eye" especially when looking about. Also, there is now a loss of vision. A quick visual acuity test does confirm the patient's own observation. And the swinging penlight test also reveals an afferent pupillary defect. Ophthalmoscopy, however, shows a normal optic disc. You are entertaining a diagnosis of optic neuritis but how to confirm it in the absence of any obvious signs of disc change?

Contrast MRI, naturally:
The above is a T1-weighted coronal image of the brain after intravenous GdDTPA, a contrast agent, to provide enhancement of the lesion. And in this case, a hyperintense left optic nerve (on the right side of the image, the white spot next to the caption) suggesting a leaky optic nerve sheath resulting from, most likely, inflammation. In contrast, the right optic nerve shows no such enhancement. This clinches the diagnosis of left optic neuritis. A course of intravenous steroid treatment is then in order. Unfortunately for the patients, optic neuritis is often an early manifest of multiple sclerosis (MS):

In fact, 15 years after the onset of optic neuritis, ca 75% of women and ca 34% of men will have developed MS (see above, the hyperintense areas) which is characterized by exacerbations and remissions of motor and/or sensory functions over a period of time. Conversely, up to 50% of MS patients will develop one episode of optic neuritis. Both types of patients are seen in the eye doctor's office.

Metabolically, proton MRS of MS brain lesions shows a decrease in the NAA/Cr ratio and an increase in the Cho/Cr ratio. MRS in conjunction with MRI therefore can help distinguish early lesions that might respond to therapy from late irreversible ones. In theory, other resonances such as lipids, lactate, glutamate, and inositol, could potentially help in the staging of MS. Certainly, any successful treatment will be evidenced by the normalization of the NMR spectra.

A more specific water-suppressed proton MRS study of an MS patient (see Narayana et al. Serial proton magnetic resonance spectroscopic imaging, contrast-enhanced magnetic resonance imaging, and quantitative lesion volumetry in multiple sclerosis. Ann Neurol 1998;43:56–71) is cited here:

The spectra shown above were obtained on Days 98 (A), 147 (B), 189 (C), and 259 (D) of a longitudinal study. The volume of interest is exhibited in the MR images. The resonance at 2 ppm is NAA. (A) is the baseline. (B) and (C) reflect transient changes, chiefly a decrease in NAA. And (D) total recovery back to baseline as that in (A).

Notice in (B), the resonance at 1.32 ppm, designated as lipids in the original report, is actually lactate [to be further verified of course]. This is consistent with the general observation that lactate accumulates abnormally during necrosis, inflammation, or other tissue injury. In other words, whenever ischemia occurs, cellular metabolism is forced into anaerobic glycolysis with lactate as the principal end-product.

We will next examine the etiology and treatment of MS.

8.4 Four-dimensional NMR spectroscopy

Yes, everybody knows that: we all live in a magnetic field - the Earth. Its field strength is a mere 30-60 micro Tesla (depending on where you live). However, a large metal tube such as a submarine deep in the ocean can cause magnetic disturbances and is therefore detectable. Rumor has it that a certain U-Boat captain founded an NMR company, most likely inspired by his wartime experience evading MADs (magnetic anomaly detectors). A submarine can become electrically charged owing to the difference in the temperature of the upper and lower hull. This is enough to create a difference in salinity of the enclosing sea water, hence a dynamic electric field around "Das Boot". A stealth submarine will have to address this issue obviously. In NMR, shielding high-field magnets is a challenge even now. Incidentally, Mr Tom Clancy has much more on anti-submarine warfare in his classic "The Hunt for Red October" (which most people on Earth have already read):
Back to the issue at hand: At some point in the not so distant future, the full potential of biological NMR will finally be realized. And it will be in the form of 4-dimensional (three spatial and one chemical-shift) NMR spectroscopy. In other words, imaging of, not just the water-protons, but the far more informative metabolites. The limiting factor is, yep, you've guessed it, the magnetic field strength.

Now picture yourself inside a magnet with a set of antennas enclosing your head. And after a certain period of rf irradiation at proton Larmor frequency and FID signal collection and processing, you are ready for the result. And it'll be a spectrum with only a large water peak. That won't be so interesting at all, will it. So, how is an MR image constructed?

Very simple, the FIDs must be encoded in three dimensions. First, along the direction of the north-south direction (the z-axis) of the magnetic field, a field gradient (i.e., the z-gradient) is applied. This gradient causes the Larmor frequency to change linearly, and a slice is selected by choosing the rf frequency corresponding to the Larmor frequency of the center of that slice. For this slice, a preparation gradient, the y-gradient, is then applied for frequency encoding, followed by the x-gradient for phase-encoding. Finally, a 2D Fourier Transform is used to transform the encoded image into the spatial domain. Why the need for a slice? Answer: to make sure enough signals are generated. A single layer of nuclei will have almost negligible signals to work with. [Note: For more information on important aspects of MRI, we recommend this site: http://mritechnicianschools.net].

Present-day clinical MRI is mostly imaging of water-protons. For the ultimate 4-dimensional NMR spectroscopy, an example is shown below:

Again, using an old standby: a rabbit lens (10-mm diameter and 5-mm thickness) incubated for 28 hours in high glucose for maximal sorbitol production. ¹³C1-glucose was used as the substrate. And NMR experiments were performed using a 9.4-Tesla spectrometer.

Part 1. MR microscopy:

Top: Schematic diagrams of the lens. The two primary regions are the cortex and the nucleus. (Left): section through the anterior-posterior poles and (Right): section through the equatorial plane.

Bottom: A water-proton image, through the equatorial plane, acquired with a spin-echo pulse sequence with TE (echo time)/TR (repetition time) = 3.88/1000ms, a 126x128 matrix, and 32 averages. Image resolution was 40 x 40 x 500 micron.

Part 2. Chemical-shift ¹³C microscopy:

At the bottom is a ¹³C spectrum acquired at 100.61MHz without proton decoupling - hence the multiplets of the biochemicals.

And the top shows color-coded images of glucose (red), sorbitol (yellow), and lactate (purple), respectively. The images were acquired with spin-echo of TE/TR = 9/500ms over 8 hours. A narrow bandwidth selective pulse (4KHz) was used with z-gradient = 800Hz/mm (slice thickness = 5mm). All three metabolites resonated within this bandwidth. The in-plane resolution was 600 x 600 micron. From the images, it is clear that there is a glucose concentration gradient with higher glucose in the cortex. And in contrast, sorbitol appears to re-distribute into the nucleus even though it is produced mostly in the cortex.

Obviously the above still cannot be done on humans. However, 7-Tesla clinical imagers are now coming on line. It is really only a matter of time before the manufacturers start seeking FDA approval for these imagers. For now, an alternative is to collect NMR spectra from, e.g., selected areas of the brain. It is possible to acquire somewhat low-resolution ³¹P, water-suppressed proton, and even ¹³C spectra at 1.5 Tesla - the most common magnet field strength used in a clinical setting today.

(Source: http://www.ajnr.org/cgi/content/full/23/8/1387)

In the above example, Cho (choline), Cr (creatine), and NAA (N-acetyl aspartate) are clearly seen. Each spectrum is from a volume of interest of 100 x 80 x 15 mm with an acquisition time of 60 min. [Note: A little long, but not intolerable from personal experience. Despite the loud knocking noises from turning gradients, some are known to doze off in the magnet.]

So, here is the current situation: MRI using 1.5-Tesla body scanners has matured as a routine clinical procedure. However, to see the metabolites, the only practical approach is to acquire NMR spectra from a volume of interest in the brain (or other body parts). This is in-vivo NMR spectroscopy, also known as MRS (magnetic resonance spectroscopy). Again, with ultra-high-field clinical magnets now rapidly becoming available, we should expect high-resolution MRS and perhaps even chemical-shift imaging soon.

For the eye, we will need to make some adjustment. Instead of the body or the head coil, we use a home-made surface coil that transmits and receives rf. Typically, it is a single-turn 1-inch coil that covers the whole eyeglobe:
It is then possible to obtain high-resolution MR images of the eye and water-suppressed proton spectra of the vitreous. Again, for the time being.

8.3 For the first time in human history

(A 4.7-Tesla horizontal-bore superconducting magnet for small animals)

Chemists have long used NMR spectroscopy to study the structure of chemicals. Usually (1) the samples are Pasteur-pipetted into a small diameter NMR tube (e.g., 5-mm or less), and (2) to improve S/N, the acquisition time is deliberately long to allow more signal averages. For the biochemists, the only possible application is to analyze PCA (perchloric acid) tissue extracts (through a procedure that requires a mortar and pestle and loads of liquid nitrogen and elbow grease). Studies of living tissues are obviously out of the question; although even PCA extracts can provide interesting NMR spectra.

It is unclear who first convinced manufacturers of NMR spectrometers that size did matter and that many biological applications were still waiting in the wings. By the mid-1980s, there was a proliferation of wide-bore high-field superconducting magnets, both vertical and horizontal. All of a sudden, in-vivo and ex-vivo experiments can now be performed. With the additional bore space, simple perfusion systems can be integrated that can maintain cell/tissue/organ viability inside the magnet. And anesthetized small animals, such as mice, rats, and rabbits, can be comfortably fitted into the bore of, e.g., a 4.7-Tesla magnet (see image at top). In time, spectrometers capable of imaging also become available thus completing the entire application of biological NMR. And whatever happened to the NMR jocks (those within our circle, of course)? As far as we know, some are still shimming (bigger and better) magnets, while others become neuroradiologists, and at least four are practicing ophthalmologists.

For eye doctors who happen to be interested in diabetic cataract formation, the 10-mm probe is ideal as it allows studies of intact rabbit lenses which coincidentally are each around 10mm in diameter. Or, 10-12 rat lenses can be grouped together and regarded as one lens. These lenses can be placed in a 10-mm NMR tube filled with incubating medium and they will stay viable during signal acquisition. Further, the medium can be prepared with normal (5.5mM) or high (35.5mM) glucose (or other sugar substrates), the latter to simulate hyperglycemia.

NMR spectroscopy not only confirms old findings but often provides new information. The latter is akin to witnessing the discovery of something new "for the first time in human history". Some examples are shown below:
The above is a ³¹P spectrum of normal rat lenses (age of the animals: 8-10 months). It shows low levels of S3P (sorbitol-3-phosphate), F3P (fructose-3-phosphate), and αGP (α-glycerophosphate), and relatively high levels of PCh (phosphorylcholine), Pi (inorganic phosphate), GPC (glycerophosphorylcholine), and ATP.

One week after diabetic induction (by using streptozotocin), there is a huge increase of S3P, F3P, and αGP:

And if the the diabetic rats are treated with an oral AR (aldose reductase) inhibitor to stop sorbitol production, then the spectrum normalizes. In fact, both S3P and F3P now disappear entirely.

S3P and F3P are both novel metabolites never seen before. In fact, to this day, we still don't know their roles in diabetic cataractogenesis. They are also present in the red cells of human diabetics for reasons unknown.

In addition, in rat lenses incubated in high glucose, there is production of sorbitol (and some fructose) with time as evidenced by the following ¹³C spectra (top: one-month-old rat lenses and bottom, old rat lenses):
On the other hand, no mannitol is produced in lenses incubated in high mannose:
Does this mean that mannose can be used as a sweetener for the diabetics? Perhaps. Mannose, in fact, does taste quite sweet. Although, as they often say: more studies are needed.

Structurally, mannose and glucose differ only in C-2, different enough for AR not to recognize and metabolize it:
Of course, there are numerous other ocular diseases that can be examined with NMR. For example, the much dreaded bacterial endophthalmitis after cataract surgery. In rabbits, intraocular Streptococcus pneumoniae caused appearance of lipids in the vitreous, suggesting a breakdown in the blood-ocular barrier:

On the left are proton spectra of the normal eye/vitreous dominated by the lactate resonance (at 1.35ppm). The infected eye/vitreous shows, in addition to lactate, methylene (1.15ppm) and methyl (0.83ppm) groups of lipids, over a span of 2-4 days. By the 10th day, the eye becomes phthitic.

Here we should point out that the concentration of water is 55.6M. Such a high concentration is great for MRI. In NMR spectroscopy, however, water-protons easily overwhelm other proton resonances. For the investigation of metabolites with a concentration in the mM range, water-suppression is therefore necessary. The above spectra were acquired by pre-saturating water signals, one of the half a dozen or so techniques.

So how do we go from NMR spectroscopy to MRI? Very simple, it is all in spatial encoding. To illustrate this point, we shall next examine MR microscopy and chemical-shift imaging.

8.2 NMR

One wonders if both Felix Purcell (1912-1997) and Edward Bloch (1905-1983) had experimented with magnets (see above) when they were little. They did, however, share the Nobel Prize in Physics in 1952 for independently creating experiments for the observation of NMR (nuclear magnetic resonance).

The growth of NMR research especially in the past two decades is nothing short of spectacular. It is a field driven entirely by the magnetic field strength (B₀) which determines the operational frequency or Lamor frequency (_ω0) of the NMR nuclei. The equation central to NMR research is this:

ω₀ = γB₀

γ is of course the immutable gyromagnetic ratio (or magnetogyro ratio - depending on whether you are a physicist or a chemist) unique to each NMR nucleus. The table below is a quick glance of the Larmor Frequency at a field strength of 1 Tesla (last column on the right):

Particle	Spin	wLarmor/B s-1T-1	n/B
Electron	1/2	1.7608 x 1011	28.025 GHz/T
Proton	1/2	2.6753 x 108	42.5781 MHz/T
Deuteron	1	0.4107 x 108	6.5357 MHz/T
Neutron	1/2	1.8326 x 108	29.1667 MHz/T
23Na	3/2	0.7076 x 108	11.2618 MHz/T
31P	1/2	1.0829 x 108	17.2349 MHz/T
14N	1	0.1935 x 108	3.08 MHz/T
13C	1/2	0.6729 x 108	10.71 MHz/T
19F	1/2	2.518 x 108	40.08 MHz/T

(Note: the study of electron spin is ESR - Electron Spin Resonance or now known as EPR - Electron Paramagnetic Resonance - an entirely different ballgame from NMR)

In the old days, field strength was represented by proton frequency. For example: what was a 400MHz (or megacycle) magnet is now known as a (400/42.5781=)9.4 Tesla magnet. Because of the notoriously low sensitivity of NMR nuclei, much improvement in signal generation relies on increasing the magnetic field strength. The higher the field strength, the more the signal intensity (or higher signal-to-noise ratio). For example, for proton, an increase from 300 to 400MHz yields an improvement in S/N of (400/300)² = 1.78 fold. Building ultra-high-field magnets is a competitive sport for the manufacturers of course.

What are these NMR nuclei? Well, any nucleus with an odd number of protons or neutrons (or combination) qualifies; for example, biologically relevant nuclei include ¹³C, ³¹P, ¹H (proton), ²³Na, ¹⁷O, ¹⁵N, ¹⁹F, and deuterium (a nucleus with spin number I=1). Of these, ³¹P and ²³Na are both 100%, and ¹H 99.98% naturally abundant. The others are only a fraction of around 1% or less and are usually used as labels; for example, ¹³C can be labeled at either C-1 or C-6 position of glucose and the glycolytic products can thus be traced. On the other hand, proton is part of the ubiquitous water molecule, and ³¹P that of ATP and other organophosphates.

All these nuclei when placed in a magnetic field will precess at certain ω₀: depending on the γ of each nucleus, as mentioned above:

(From: teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm)

If a radiofrequency (rf) perpendicular to the north-south direction, or conventionally, the z-axis of the magnetic field is applied at the frequency of a nucleus of interest, then this nucleus will be tipped into the x-y plane. When the rf is turned off, the nucleus will begin to return to its original ground state through a process, known as relaxation. Relaxation is defined by longitudinal relaxation time (T1) and transverse relaxation time (T2). And the signals produced in the rf field are known as the FIDs (free induction decays) which can be picked up by an antenna and processed (through Fourier Transform from time into frequency domain) to generate NMR spectra.

(Note: No Bruker share-holders here)

The above is a typical high-field vertical-bore superconducting magnet used in NMR spectroscopy. It requires periodic feeding of liquid nitrogen and liquid helium. A probe is inserted from the bottom and the sample (in an NMR tube) loaded from the top, controlled by compressed air. A computer console is situated nearby for shimming, pulse sequence delivery, signal collection, spectral generation and printing.

An NMR probe is the heart of the spectrometer, it fits vertically into the bore of the magnet. They come in different bore sizes as well, e.g., 2, 5, 10, and 20mm diameters are common. These probes can be broadband or single frequency.

Before insertion, it needs to be optimized by using tuning and matching rods with the aid of an oscilloscope. Tuning is simply to make sure the probe is set to the frequency of interest. And matching is to ensure the impedance is equal to the external electronic circuitry so that maximal power can be transferred. These two operations are actually inter-related:

Shimming is an art, its purpose is to maximize the magnetic field homogeneity which greatly affects spectral resolution. Some "NMR jocks" are known to take extraordinarily long time (e.g., forever) to shim the magnet - to perfection. And a lot of them know how to read the FIDs even before FT. The resulting spectra are often strikingly well-resolved. NMR jocks are a special breed. Much like computer hackers, NMR jocks also survive on pizza and coke, except the food and drink are paid for with cash, as their credit cards are usually erased by the magnets. (Try and explain this to the pizzeria owners.)

Next, we will examine a few examples of NMR spectroscopy of ocular tissues - before entering the field of MRI (magnetic resonance imaging).

8.1 Brain is an extension of the eyes

(The visual pathway - looking from the base of the brain)

The visual pathway of course travels all the way from the eyes to the back of the brain (see above, in red). Anything untoward that occurs along the pathway will manifest as an ophthalmic problem. And in addition to the second cranial nerve, the extraocular muscles are innervated by the third, 4th and 6th cranial nerves. Plus, the pupillary responses are controlled by yet another set of nervous pathway. Neuro-ophthalmology is indeed a very interesting and challenging field. With very simple tools, such as a penlight, an occluder, a tangent screen and/or perimeter, definitive diagnosis often can be reached. With time, more tools become available. And brain imaging is now consisted of many modalities:

(From socialfiction.org)

These modalities include the old standby CT, the re-vamped PET, the anatomical MRI and the more powerful fMRI (f=functional), and a close relative of fMRI, the Optical Imaging of Intrinsic Signals (OIS). (Note: Cryo is imaging of frozen brain in slices, not for the living.)

Of these, we will go into more about MRI, in order to discuss important subjects such as "perivenous syndrome" and "brain plasticity". And to understand MRI, we will need to start from its humble beginning, NMR. Next post is therefore "8.2 NMR".