Exploration of Human Brain Tissue

Gregor T. Overney, Ph.D.
Sunnyvale, California, USA

Fibrous Astrocytes (Objective 40x)

Introduction

The human brain is the most wonderful system known to us. It is the control center for all our thoughts and senses and allows us to interact with our universe in magnificent ways. In this short article, I explore a few microscopic details of the human brain. For the reader unfamiliar with histology, I suggest reading a recently published article entitled "Human Histology for Amateur Microscopists" [1].

After a short introduction into the anatomy of the human brain, I will look at large neurons, called Purkinje cells, which are located in the cerebellum of the human brain, followed by an exploration of astrocytes, which have been made visible with the famous Cajal/Golgi heavy metal impregnation.

Anatomy of the Human Brain

The brain is the most complex organ of our central nervous system (CNS). It is composed of neurons, blood vessels, and glial cells (supporting cells of the CNS). The amount of neurons in the human brain is estimated to be around 10 billion interconnected neurons. Each of these neurons is connected to thousand neighboring neurons. A neuron has one cell body, containing the nucleus and surrounding cytoplasm (perikaryon), one axon (up to one meter in length), and one or more dendrites, which extend from the cell body. Dendrites are highly branched and form a so called dendritic tree. The axon splits into several branches. Each of them ends in so called terminal buttons. Those terminal buttons exchange signals over the synaptic gap via neurotransmitters to dendrites of a neighboring dendritic tree belonging to an other neuron. (See Fig. 1 for a simple drawing of two neurons.)

The gray matter of the CNS contains most of the neuron cell bodies, while the white matter of the CNS contains the axons. The white appearance of the fresh tissue is caused by lipid in the myelin sheaths of the axons. Myelin is 80% lipid and 20% protein and formed by oligodendrocytes (type of neuroglia). Myelin sheaths are used to insulate axons. (BTW, a nerve cell with a myelin sheath around its axon is called myelinated.)

The human brain consists of various major parts, each fulfilling highly complex functions. These parts are paired cerebral hemispheres, which are invested by the cerebral cortex, diencephalon, which is formed by large gray matter structures, midbrain, pons, medulla, and cerebellum. (See Fig. 2 for an overview drawing of a human brain.) The cerebellum is responsible for coordinating muscular activity and maintains posture and equilibrium.

In the following paragraphs, we look at the microscopic structure of two selected features of the human brain, Purkinje cells in the cerebellum and one type of glial cells of the white matter, called fibrous astrocytes.

Purkinje Cells

We are looking at the cerebellum of the human brain. The cerebellum consists of a cortex of gray matter (outer layers) and a central core of white matter. Located in the cerebellum are large, distinct neurons known as Purkinje cells. A Purkinje cell in the cerebellum has a single small axon at one pole and an extraordinary, finely branching dendritic tree at the other pole. - Fig. 3a shows a section of the cerebellum using a 4x plan achromat objective. (A company called MICR-O-SLIDE makes this slide. Unfortunately, I am not familiar with the staining technique used; it might be a trichrome stain.) We clearly recognize three distinct layers (see Fig. 3a). The cerebellar cortex (made out of gray matter) consists of a series of deeply convoluted folds, called folia, and divides into the granular layer, which is extremely cellular, and the molecular layer, which contains relatively few neurons and large numbers of unmyelinated fibres. The innermost layer (3rd layer) is white matter. The Purkinje cells, forming the so-called Purkinje cell layer, are between the granular and molecular layer. Fig. 3b and Fig. 3c show more details. The reader can easily recognize the very large cell bodies of the Purkinje cells. Purkinje cells have a relatively fine axon that is extending down trough the granular layer (difficult to see with this slide). Their extensively branching dendritic tree arborises into the molecular layer. The dendritic tree is not visible with the staining technique used for this slide. To make this tree visible, a different staining technique can be used, which is known as heavy metal impregnation. We will learn more about this staining technique in the next chapter. At time of writing this article, I had no access to a metal impregnated tissue sample that shows Purkinje cells.

Glial Cells - Neuroglia

How are neurons of the CNS held in place and how are their axons electrically insulated to avoid "short circuiting neurons"? Specialized cells in the human brain, called glial cells or neuroglia, provide most of these functions of protection, insulation, and physical support. There are three types of glial cells, astrocytes, oligodendrocytes, and microglia. The following table summarizes the functions of glial cells.

Type of CNS Supporting Cells (Glial Cells)	Functions
Astrocytes	Provide physical support to neurons Clean up carcasses of dead neurons Provide controlling chemicals to neurons Play important role in providing nourishment to neurons
Oligodendrocytes	Provide physical support to neurons Produce myelin sheath that insulates axons
Microglia	Clean up CNS debris Play role of immune system in brain (similar to microphages in blood)

Let us focus on astrocytes (star shaped cells). Astrocytes provide mechanical support and mediate the exchange of metabolites between neurons and blood vessels. Astrocytes are not the blood-brain barrier. The blood-brain barrier is a protective barrier that works like a filter. It lets certain molecules pass while other substances of the blood are prevented from entering brain tissue. The blood-brain barrier is formed by nothing more than endothelial cells (cells that form capillaries). Astrocytes induce these endothelial cells to form tight junctions with each other.

Astrocytes couple with blood vessels through so-called footplates, which are specialized attachments to blood vessels. There are two different types of astrocytes. In the gray matter, we can find protoplasmic astrocytes. These astrocytes have many short, relatively thick processes. The astrocytes of the white matter, called fibrous astrocytes, are less branched but their processes radiate from the cell body for considerable distance. - To the best of my knowledge, we will be looking at fibrous astrocytes, which are located in the white matter [2].

Before exploring these interesting cells, I want to mention a few details about a powerful staining technique used to make astrocytes, as well as neurons, visible. This technique is called heavy metal impregnation. In 1873, Camillo Golgi (1843 - 1926) published a short note in the Gazzetta Medica Italiana about the structure of the gray matter in the brain. He described his success in observing the elements of the nervous tissue after subjecting brain tissue samples to metallic impregnation. The black reaction, Golgi discovered, is based on nervous tissue hardening in potassium bichromate and impregnation with silver nitrate. But Golgi did not correctly describe the microscopic structure of the nervous system. In 1887, Santiago Ramóny Cajal (1852 - 1934) was introduced to the Golgi method. With this method, Cajal was able to correctly describe the nervous system. In 1909, Cajal published his findings in the classic text 'Histologie du système nerveux de l'homme et des vertébrés'. In 1906, Cajal and Golgi shared the Nobel Price for their study of the nervous system.

For the heavy metal impregnation technique, thick sections should be used to increase the likelihood of having complete neurons being included in the plane of the section. The technical details of the metal impregnation are not discussed in this article. The reader may read Cajal's standard text [3]. - I was able to purchase slide 93 W 6322 from WARD [4] labeled 'Astrocytes (sect.)' showing astrocytes highlighted by Cajal's gold chloride method. Fig. 4 shows some clearly visible astrocytes (objective 40x). The reader can easily see a network formed by these astrocytes. In the region examined, I was not able to find neuron cell bodies and hence conclude that the region depicted in Fig. 4 belongs to white matter. A blood vessel is also visible in Fig. 4 (indicated by 'B').

BTW, serious lack of contrast forced me to "upgrade" the digital camera equipment from a Nikon Coolpix 995 to a more specialized CCD camera using a cooled CCD. Although a Nikon Coolpix 995 is a very suitable choice for most bright-field applications of H&E stained histology slides, I could not obtain sufficient contrast for this application. For comparison, Fig. 5 shows an image of astrocytes from slide 93 W 6322, which has been recorded with a Coolpix 995. In Fig. 4, the reader can find tiny circles that are caused by this specialized CCD camera. This is an artifact that cannot be observed with the Coolpix camera.

Summary and Acknowledgements

We looked at Purkinje cells of the cerebellum of the human brain and investigated the network of astrocytes, which are important supporting cells of the central nervous system (CNS). For this work, I used Wheater’s histology text and color atlas [5].

I want to thank Dr. Fei Liu for many suggestions and stimulating discussions. The technical support of C&N for designing the HTML version of this article is greatly acknowledged. Last but not least, I thank all anonymous supporters who provided assistance and equipment.

Comments to the author, Gregor Overney, are welcomed.

References

[1]	G. Overney. Human Histology for Amateur Microscopists, Micscape Magazine 82 (2002), and references therein.
[2]	On page 135 in [5], one picture of astrocytes is shown using the Cajal method (Fig. 7.21 b). Unfortunately, the authors leave it up to the reader to identify the type of the astrocytes depicted.
[3]	S. R. Cajal. Histology of the Nervous System of Man and Vertebrates (translated N. Swanson and L. W. Swanson), Oxford University Press, New York (1995); S. R. Cajal. Texture of the Nervous System of Man and the Vertebrates (annotated and edited translation of original Spanish text by P. Pasik and T. Pasik), Springer Verlag, New York (2000).
[4]	WARD’s Natural Science Establishment, POB 92912, Rochester, NY 14642.
[5]	B. Young, J. W. Heath. Wheater’s Functional Histology, 4th Edition, Churchill Livingstone, London (2001).
[6]	Background adjustment (or subtraction) can be performed using either the built-in feature of the Nikon Coolpix camera or with the help of image processing software. For instance with Paint Shop Pro 7.04, using the menu "Colors->Adjust->Levels", the user can easily flatten the color spectrum with respect to any RGB color-value. Of course, post-processed background adjustment can reduce the dynamic range per color channel. For 24-bit RGB, the maximum color range per color channel is 8-bit, or 256 distinct values, per channel.

Figure Captions and Figures

The setup for photomicrography consisted of a bright-field microscope Nikon Eclipse E200-F with Köhler illumination, Nikon CFI60 E-Plan Achromat (4x and 10x) and Plan Fluor (40x) objectives. For Fig. 3 (a, b and c) and Fig. 5, I used a Nikon Coolpix 995 digital camera with a Nikon MDC2 relay lens. For Fig. 4, I used a cooled, high contrast CCD camera with a RGB color-wheel. All photomicrographs were image processed. A filter to sharpen the image was applied and background subtraction was performed to increase contrast [6]. Text and graphics objects (such as arrows and dashed lines) were added using Paint Shop Pro 7 from JASC (see http://www.jasc.com/).

Fig. 1: Simple drawing of two interacting neurons. This is only a sketch and does not represent actual neurons of the human brain. (Click on image for larger version.)

Fig. 2: Overview drawing of human brain. (Click on image for larger version.)

Fig. 3: All images show a section of the cerebellum of the human brain. Fig. 3a is an overview of all layers making up the cerebellum, such as cerebellar cortex and supporting white matter. Fig. 3b and Fig. 3c show more details of the Purkinje cell layer. The objective used for Fig. 3b is 10x and the one used for Fig. 3c is 40x. (Click on images for larger versions.)

Fig. 4: High contrast image of fibrous astrocytes (type of glial cells located in white matter) in human brain using cooled CCD camera (objective 40x). Two of the astrocytes are labeled with letter A. On the right side there is a blood vessel visible (labeled with letter B). (Click on image for larger version.)