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Thursday, December 3, 2009

H.M.'s Brain

The philosopher Martin Heidegger reasoned in his treatise "Sein und Zeit" that our sense of time confers our sense of being (Heidegger, 1976). Henry Gustav Molaison could attest to that. In order to relieve Henry from severe bouts of epileptic seizures, a part of his temporal lobes had been removed when he was in his late twenties.

The seizures subsided. However, as an unwanted consequence of the surgery, he had lost essential elements of his memory. He could not remember who he was and had to discover his identity anew every day. One day late in his adult life, Henry was caught curiously examining his face in a mirror. He called out in amazement, "I am not a boy!" In Heidegger's words, Henry Molaison had lost his sense of time and, with that, his sense of being.

As patient H.M., Henry volunteered for scientific research throughout his life, becoming one of the most intensely examined cases in psychology. He willed his brain to research. Yesterday a year ago, he passed away of respiratory complications at the age of 82. On the first anniversary of his passing, the histological processing of his brain was begun at the University of California San Diego. The university's Brain Observatory provides live online footage of the sectioning of the frozen whole brain on a cryotome until Dec. 4, 2009. The brain is cut coronally, that is in the transverse plane. Seventy micrometer-thin sections are collected for histology at equally-spaced intervals. At the speed maintained thus far, we shall reach the site of surgery tomorrow.

The ensuing histological examination of his brain will complete Henry's scientific journey as he willed. The findings may provide important insights into the histochemistry of memory, constituting the keystone in the arch of knowledge that H.M. allowed us to build.

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Addenda
  • At 5:00 am (CST), we are in the midst of the temporal lobe of H.M.'s brain. If you look closely at the bottom of the brain (ventral, on the left hand side of the observer), you see cauliflower-shaped protrusions on both sides. These are the temporal lobes. There is a void inside the structures. Parts of the hippocampus seem absent (12/04/09).
  • At about noon, in section 1672 (46,344 micrometers), we can see a cavity in left lingual gyrus (in the hemisphere closest to the observer) (12/04/09).
  • At 5:00 pm (CST), we are entering the occipital lobes; at 10:00 pm we passed the middle of the occipital lobes (12/04/09). 
  • We should be able to detect signs of nerve cell and nerve fiber degeneration in the brain regions that were connected to the brain tissue that was removed or damaged otherwise during surgery, particularly in the structures of the limbic system. Degenerating nerve fibers can be rendered decades after the damage and ameboid microglia may reside in the affected locations (Miklossy and others, 1991). According to von Monakow's concept of diaschisis, the affected regions can be far-flung and distant from the site of immediate damage, comprising the opposite cerebral and cerebellar hemispheres. That is why H.M.'s whole brain needed to be sectioned, and 2,401 sections were collected (12/05/09).
  • The footage covering the sectioning does not seem to be available on The Brain Observatory's site at this time. You may find a CBS report broadcast Dec. 3, 2009, instructive (01/01/10):
  • Brain sections are prepared for histology with two distinctly different methods. One way requires that the tissue is embedded in hot paraffin wax or plastic. The tissue is sectioned at room temperature on a bench top with a microtome or ultramicrotome, respectively. Paraffin embedding is widely used in routine pathology. Plastic is used to embed specimen to be cut into ultra-thin sections for electron microscopy. The other method of tissue preparation requires that the specimen are frozen. Frozen tissue has to be stored and sectioned at temperatures below 0 ℃.

    With either method of preparation, the tissue is commonly first soaked in solutions of formaldehyde, glutaraldehyde or a mixture of both. The aldehydes help bond cellular protein, preserving cellular structure during the further processing steps. H.M.'s brain was then soaked in highly concentrated sucrose solutions and embedded in dextran. Sucrose and dextran are sugars. The sucrose prevents ice crystals from forming in the cells when the tissue is frozen. The growing crystals would otherwise burst the cells. The dextran helps preserve the integrity of the sections, preventing tissue from breaking off the perimeter of the specimen during cutting. Once the brain is fixed and cryo-protected, it can be cut into sections that are only tens of micrometers thick on a microtome on which only the stage holding the brain is kept at subzero temperatures. The sections are picked up from the microtome's knife with a brush and can be processed freely floating in solutions containing the substances needed for the chosen histological staining procedure.

    There are numerous histological staining procedures. Most comprise chemical reactions. Others involve antibodies that bind specifically to cellular proteins or fluorescent dyes that fill particular cellular components. Among the oldest procedures is the silver impregnation method named after  Camillo Golgi, rendering the cell bodies of nerve cells and a good part of their processes (dendrites) and protrusions (dendritic spines). This method helped Ramón Cajal to divide the cerebral cortex into six layers, a distinction that is still used today. Golgi and Cajal were awarded the Nobel Prize for their contributions. The most commonly employed methods, however, are the Nissl stain for cell bodies and the myelin stain that stains the fatty sheeth around nerve cell fibers in the brain's white matter. These methods will certainly be used with some sections of H.M.'s brain. After staining, the sections are mounted on glass slides, dehydrated in solutions of increasing alcohol concentration, embedded in a clear organic solvent-based glue and covered with thin glass coverslips.

    The methods of tissue preparation described above cannot be used when the procedures require that fixation-sensitive cellular proteins remain functional or when water-soluble, diffusible substances are to be localized in the tissue. In that case, the tissue must remain unfixed and immersion in sucrose cannot be used. The tissue has to be shock frozen at -70 ℃ and lower, and the sections must be kept frozen during sectioning. To fulfill this need the whole microtome is encased in a refrigerated cabinet. The instrument is then called a cryostat. Ten or 20 micrometer-thick sections are cut and picked up on chilled glass slides and quickly dried on a hotplate outside the cabinet. The subsequent procedures must consequently be carried out on the slide-mounted sections. This method is used for the autoradiography of diffusible radioactively-labeled tracers. For autoradiography, the sections are exposed to x-ray films or radiation-sensitive semiconductor chips that produce gray-scale images of the distribution of the tracer in the sections. The optical densities in the images called autoradiograms are proportional to the concentration of the tracer in the tissue and can be measured.

    In order to trace metabolites in whole bodies, Sven Ullberg and colleagues at Uppsala University, Sweden, engineered a microtome sufficiently sturdy to cut thin sections through the whole body of small animals. The microtome was placed it in a chest freezer with windows in the lid (Larsson and Ullberg, 1981). The instrument is called a whole-body cryotome and was commercially available from the Swedish company LKB. I was privileged to use a LKB cryotome 30 years ago for functional imaging studies (Melzer, 1984). The picture below shows a whole rat embedded length-wise in dextran and frozen in liquid nitrogen at -173 ℃. The dextran block with the rat is mounted on the sliding stage of the cryotome for sectioning.
    The block of dextran with the rat resembles that with H.M.'s brain in the footage of the Brain Observatory which misled me initially. But as I pointed out, H.M.'s brain was fixed and protected with sucrose. Only the microtome stage had to be cooled. No freezer chest was needed. Jacopo's hands stayed warm.

    One great advantage of whole body sectioning is that the sections render the organs in their actual position inside the body. The picture below shows a the block surface of the head of a sandrat embedded in dextran and cut transversely at the level of the midbrain.
    The structure at the center is the brain. The glassy cavities on both sides of the brain are the inner and middle ears. The middle ear cavities of sandrats are remarkably enlarged compared with other rodents, serving as a low pass filter. The animal had been exposed to sound for functional imaging. Brain activation images are shown in my post entitled "The Quest for the Infrasound Acoustic Fovea" and dated Oct. 12, 2009.

    Since the brain was preserved accurately as it was situated in the scull, we were able to pinpoint brain structures with stereotaxic precision, a goal that may be achieved with structural magnetic resonance imaging today. That method did not exist then.

    In addition to functional imaging, fresh tissue tissue sections permit us to examine the distribution of radioactively-labeled ligands that bind to specific receptors on cell surface or probes that identify sequences of genetic code. These procedures can not be applied to the sections of H.M.'s brain. However, even with the histological techniques available a treasure of knowledge will be uncovered from the brain that Harvey Molaison kindly willed.

    In the past three decades most departments of anatomy have been transformed into departments of cell biology for good reason. However, gazing through a microscope at the multitude of diversely shaped nerve cells in a Golgi-stained section through cerebral cortex still remains as fascinating as gazing through a telescope at the myriad of stars in the night sky. The neuroanatomical methods that make this experience possible resemble in many ways forms of art, though rather of an artisan than of an artist. Perhaps, the work on Harvey's brain will rekindle interest in this type of expertise, paving the way for a renaissance of neuroanatomy (01/02/10).
  • The Allen Institute for Brain Science in Seattle, WA, compiled the first comprehensive atlas of local gene expression in the mouse brain five years ago and has now released a similar atlas for the human brain (04/13/2011):

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Thursday, November 19, 2009

Advances in Spongiform Encephalopathy

Well into the 1970s, members of the Fore tribe of Papua New Guinea were afflicted by a devastating neuro-degenerative epidemic known as kuru. The disease shares considerable similarity with spongiform encephalopathy first described by the German physician/scientists Hans Gerhard Creutzfeldt and Alfons Maria Jakob in the 1920s, CJD for short. Often more than a decade after infection, the afflicted develop conspicuous trembling and severe motor dysfunction as a consequence of progressive nerve cell death in the central nervous system. The video below portrays the symptoms of this horrible disease graphically. Some readers may find the footage disconcerting.


As a consequence of nerve cell degeneration, the brain tissue shrinks in places. Microscopic holes pockmark the tissue like a sponge in histological preparations.

Kuru may have been acquired through the inheritance of defective genes. Isolated tight-knit island communities like the tribes of Papua New Guinea are known to be particularly vulnerable to inheritable genetic defects because of their limited gene pools. Indeed, genetic mutations seem to cause spongiform encephalopathy. Recently, Wang and others (2008) provided evidence for the underlying molecular mechanisms.

Peculiarly, among the Fore of Papua New Guinea particularly women and children developed kuru. The Fore were known to ingest the bodies of deceased loved ones during funeral rites in order to capture their life forces. Women and children ate mainly brain parts. Perhaps the origin of the disease was passed along with the brain tissue. In support of this idea, the late American Nobel Prize-laureate Daniel Carleton Gajdusek proved that kuru was indeed an infectious disease that could be transmitted even to other primates. Curtailing cannibalism on the islands would drastically reduce the occurrence of the disease.

Years later, Stanley Prusiner would win the Nobel Prize for ultimately identifying the molecular basis of the transmitted variant of spongiform encephalopathy. The infectious agent was not an organism. Nerve cells incorporated misfolded proteins known as prions that attached to native forms of functional cellular proteins, forcing them to undergo a similar conformational change. Like a snowball, the dysfunctional protein grows forming amyloid plaques that eventually destroy the cells.

Misfolded prions are fairly stable, temperature resistant molecules, remaining active even in soil outdoors for decades. Scrapies, which causes spongiform encephalopathy among sheep and goats, is believed to be contracted through fodder contaminated with urine and feces from infected animals.  Bovine spongiform encephalopathy (BSE), popularly known as Mad Cow Disease, has been shown to be transmitted by protein-enriched power feed contaminated with prions. Beef from infected cattle may cause CJD in people.

Remarkably, however, some highly-exposed Fore remained unaffected by kuru. In this week's issue of the New England Journal of Medicine, researchers at University College of London report that they found a new variant of the gene PRNP encoding prion protein that is unique to the resilient tribal members and may improve resistance to the disease (Mead and others, 2009). The newly discovered genetic variant, called G127V, appears to render the native prion protein less pliable to conformational change induced by misfolded protein. It was detected in half of the women who were homozygotes for an already known resilience factor. That is, they possessed identical variants of PRNP on both chromosomes carrying the gene. By contrast, tribespeople who succombed to kuru did not possess this variant, and it is absent from the global human population unexposed to the disease.

This discovery does not only open new avenues for the treatment of spongiform encephalopathy, but moreover further affirms genotypal variation and phenotypal selection as fundamental evolutionary forces that affect all living things as Charles Darwin proposed in his theory published in "On the Origin of Species" almost to the day 150 years ago. Darwin would have been delighted to learn about this finding, particularly in the year of his 200th birthday.

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Wednesday, November 4, 2009

Constantin von Monakow & Brain Plasticity

Constantin von Monakow was born in Bobresovo, Poland at that time, on this day in 1853.  He was to become one of the most eminent neuroanatomists, neurologists and psychiatrists of his age.  Below I attempt to highlight some of his achievements, relying mainly on Mario Wiesendanger's informative article in the Comptes Rendus Biologies (Wiesendanger, 2006).

Von Monakow spent most of his life in and near Zürich, Switzerland, where his family moved in his youth. He studied medicine, worked with Eduard Hitzig at one of the first psychiatric research clinics in continental Europe, the Burghölzli. The principal investigators at the clinic led by Auguste Forel had fully recognized the role of cerebral organization in mental illness.  Hitzig and Gustav Theodor Fritsch were among the first to localize function in the cerebral cortex with electrical stimulation electrodes.  Hitizig hired von Monakow as assistant and taught him histological methods.  At a brief visit with the famous anatomist Bernhard von Gudden in Munich, he familiarized himself further with silver impregnation methods that permitted him to visualize degenerating nerve fibers. Injury to brain tissue, precipitated by hemorrhagic bleeding, stroke or trauma, results in the degeneration of the nerve cell connections between the affected location with other parts of the brain.  Tracing degenerating nerve fibers, therefore, could be used to examine pathways among brain structures.

Von Monakows independent research would begin under small and unusual circumstances as attending physician at St. Pirminsberg, a psychiatric asylum near Bad Ragaz, a spa not far from Zürich.  One day, while inspecting the premises, he discovered a brand new microtome stowed away in an unused closet.  He knew how to use the microtome and turned the pantry into a small histological laboratory to get a series of experiments off the ground that would uncover the brain's visual pathway.  The faculty of the University of Zürich accepted the publication of the results of these experiments as qualification for the right to teach (habilitation), and he started to give lessons at the university without salary, while attempting to maintain a private practice for neurology and general medicine in the city.  The practice barely supported him. Yet he managed to continue his research in a small laboratory he sat up for himself.

Eventually in 1894, von Monakow received a call from the University of Innsbruck, Austria, to assume a chair in psychiatry.  The offer compelled the government of Zürich to make a counterproposal which von Monakow happily accepted, although the appointment was only at the level of associate professor. Despite the lower rank, the new post permitted him to continue his research in his laboratory, which became known as the Brain Institute and serve as the director of a psychiatric policlinic.  Finally, 17 years after he graduated from medical school, he had garnered a stable position, securing financial support for his research and a stable income.

Von Monakow worked full-time at the university until his retirement at the age of 70, and continued another four years as honorary professor and director of his laboratory, which became known as the Brain Institute. During his years as professor, he maintained an active journal club, enjoying regular visits from a number of eminent scientists and physicians. Among others, the fourth director of the Burghölzli, Auguste Forel, and the famed neuroanatomist and neurologist Constantin von Economo were in attendance as much as the founder of psychotherapy, Carl Gustav Jung, whose long-hidden self-analysis, known as The Red Book, has just been published. 

Constantin von Monakow passed away in 1930 at the age of 77, and his former responsibilities were divided among several successors.  In 1962, Konrad Akert succeeded in recreating Von Monakow's Brain Institute as The Institute for Brain Research, which has remained a vibrant site for world-class neuroscience research to the day. 

Constantin von Monakow was a prolific writer and published a long list of scientific articles, book chapters, books, hand books and anatomical brain atlases.  His work entitled "Die Lokalisation im Grosshirn und der Abbau der Funktion durch kortikale Herde" (Localization in the Cerebral Cortex and Loss of Function Produced by Cortical Lesions) arguably became one of his most notable contributions.  In this book, he laid out comprehensively the principles of chronogenic localization and diaschisis.

Von Monakow's observations suggested that the cortical and subcortical components of a brain pathway cooperate in precisely timed sequence to synergistically effect coordinated brain function, though the components may not necessarily be situated near each other.  He called the cooperative, yet distributed localization of brain function chronogenic localization.  His concept ran counter to the widely-held belief at the time that brain function was localized in circumscribed centers dedicated to particular tasks.  Phrenology represented the most extreme and popular variation of that idea.  History would prove von Monakow right.  The functional brain imaging studies conducted today commonly render multiple loci of activation in cerebral cortex when the participants execute a task, confirming that brain functions are indeed carried out by distributed nerve cell networks involving a number of cortical areas.

For example, using functional magnetic resonance imaging, my colleagues and I observed numerous foci of activation scattered across cerebral cortex when people with severe visual disability read Braille with their fingertips (Melzer and others, 2001). Intriguingly, cortical areas in the occipital lobes were involved that process visual information in sighted people. The animation below shows these foci in a 36 mm slice through cerebral cortex. The poles of the occipital lobes point to the bottom at the finish.


As another significant finding, von Monakow observed that acute damage to one component of a brain system immediately depressed the function of the system's unharmed components, disabling their coordinated cooperation.  He called this immediate loss of adequate control over integrated brain function diaschisis.

Von Monakow noted furthermore that with time functionality would return, though a residual deficit almost always remained. Differences in the timing of the recovery of various aspects of the lost function would bare its chronogenic localization.  The recovery would help identify each component's contribution to the integrated function.  Nerve cell plasticity would permit the unharmed components to reorganize and reintegrate. However, he could only speculate on the underlying cellular mechanisms.

Von Monakow proposed that the increased use of existing, undamaged collateral nerve cell connections within cerebral cortex stimulated the development of new connections, recruiting nerve cells in other cortical areas for the recovery of lost function. Furthermore, he recognized that exercise benefited recovery, suggesting that exercise-related stimulation facilitated the growth of new, more extensive nerve cell connections. As a consequence novel behaviors would be learned to compensate for residual deficits.

In own research, my colleagues and I observed that nerve cells in the vicinity of a stroke lesion in the cerebral cortex respond to sensory input only at short latency in the days after the infarct.  By contrast, responses at long latency were suppressed (Melzer and others, 2006). Inputs from intracortical nerve cell connections are thought to drive the long latency responses.  The observed suppression is entirely consistent with von Monakow's diaschisis.

Von Monakow's concept of distributed nerve cell networks underlying brain function was astoundingly modern.  His ideas of recovery of function were profoundly forward looking.  More than a century after their inception, his ideas are still inspiring intense research.

References
  • Melzer P, Maguire MJ, Ebner FF (2006) Rat barrel cortex as a model for stroke analysis. Soc Neurosci Abst:583.13.
  • Melzer P, Morgan VL, Pickens DR, Price RR, Wall RS, Ebner FF (2001) Cortical activation during Braille reading is influenced by early visual experience in subjects with severe visual disability: a correlational fMRI study. Hum Brain Mapp 14:186-195.
  • Monakow von C (1914) Die Lokalisation im Grosshirn und der Abbau der Funktion durch kortikale Herde. J.F. Bergmann, Wiesbaden.
  • Wiesendanger M (2006) Constantin von Monakow (1853-1930): a pioneer in interdisciplinary brain research and a humanist. C R Biol 329:406-418.
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