Assignment 2: Paper Review

The Role of Myelin in Theiler's Virus Persistence in the Central Nervous System

The Authors tried to find a link between the presence of the myelin in the persistence in the central nervous system by the Theiler’s murine encephalomyelitis virus (TMEV). They did this by comparing the wild-type mouse with the shiverer mouse. The shiverer mouse is a mutant with a deletion in the myelin basic protein gene (Mbp), they found previously that this mouse is resistant to persistent infection by TMEV. So they used this mouse to examine the importance of myelin in the persistence of the disease.

They knew from previous experiments that the shiverer mouse where susceptible to the “early disease” which lasts approximately two weeks after infection. The virus causes an acute encephalomyelitis with infection of neurons and to a lesser extent, of macrophages and astrocytes in the grey matter. From this point the virus is either cleared by the immune system or it persists, this persistence of the infection causes gait disorders and incontinence, and it is referred to as the “late disease”. They found that these shiverer mice were immune to this late disease. For this mutant, the resistance to persistent infection cannot be overcome by increasing viral dose. The extreme resistance of the shiverer mice indicated that the Mbp mutations interacted with essential steps in the pathway leading to viral persistence. Their experiments were designed to identify the step that leads to the persistence of a picornavirus in CNS white matter by focusing on the shiverer mutant.

Their first hypothesis stated that the resistance of the shiverer mice is not mediated by radiosensitive bone marrow-derived cells. They constructed immunological chimeras between the wild type and shiverer mice. The mice were lethally irradiated and their immune systems were reconstituted with autologous or heterologous bone marrow cells. Bone marrow grafted as well as control wild type and shiverer mice were inoculated intracerebrally with 106 plaque forming units of TMEV. The viral loads in the spinal cord were measured forty-five days postinoculation. The control mice showed the expected susceptibility and resistance of wild-type and shiverer mice. The shiverer mice that received wild type bone marrow cells remained resistant, and the wild-type mice that received mutant bone marrow remained susceptible. Therefore, the radiosensitive immune cells of the shiverer mice are not responsible for resistance to persistent infection. They also tested to make sure that since microglial cells, that secrete cytokines and chemokines which play an important role in the recruitment of inflammatory cells to the site of infection, where not responsible for the resistance to persistence. The microglial cells turn over very slowly, if at all, so they would not have been exchanged by hone marrow grafting. Wild-type and Shiverer mice were injected intracranially with 10ug of polyinosinic:polycytidilic acid. Inflammatory cells were extracted from the CNS after eighteen hours and analyzed by flow cytometry. The results show that the percentage of activated cells of monocytic origin was the same for both types of mice. They also compared the inflammatory cells present in the brain of wild-type and shiverer mice 5 days postinoculation with the virus. The results of flow cytometry show that there is the same frequency of each cell type in both types of mice. Therefore the shiverer mutant does not affect the activation of microglial cells, the recruitment of inflammatory cells and generally the adaptive bone marrow mediated immune response.

Their second hypothesis was that the early disease is not altered by the shiverer mutation. Their next experiment tested the possibility that the mutation impaired an important step of the viral life cycle in the CNS. The virus was present mainly in cortex, hippocampus and hypothalamus The pattern was the same for the wild-type and mutant mice. The macrophages were also tested between the two mice but no difference was found.

Their next hypothesis was that shiverer mutation does not alter the permissiveness of oligodendrocytes to TMEV. MBP is a major myelin protein, and it is expressed by the oligodendrocytes. These cells are infected in the late disease, it was considered that the mutation could alter their permissiveness to the virus. They used color immunoflueorescence to characterize the infection. Their results show that a single cycle of viral infection was achieved in half the oligodendrocytes. The differences between the wild type and mutant mice were not statistically significant.

Their fourth hypothesis was that the Myelin contains viral antigens during persistent infections. They looked at longitudinal and transverse frozen sections of the spinal cord of persistently infected mice. They found that there was capsid antigens forming linear patterns with the same longitudinal orientation of the axons. This confirmed that myelin sheaths may contain viral capsid antigens during persistent infection. They show that the linear pattern of viral RNA and antigens observed during persistent infection of white matter is due to the infection of myelin, more than to the presence of viral particles in the axons.

Their next idea was that the virus transported in the axons is the source of infection of myelin and oligodendrocytes. Since the virus infects neurons during early disease and is transported axonally, myelin might be exposed to axonally transported virus, and infection could spread secondarily to the cell body of the oligodendrocyte. Or, oligodendrocyte cell bodies could be infected first, and the infection could spread outwardly to the myelin sheaths. To distinguish between these two ideas they used the retina and the optic nerve which are important parts of the CNS. The ganglion cells of the retina which are adjacent to the vitreous chamber, send their axons caudally through the optic nerve where they are myelinated by oligodendrocytes. They thought that if ganglion cells could be infected and if the virus were transported in optic nerve axons, it should be possible to follow the spread of the virus from axons to glial cells. By infecting one eye only, the contralateral optic nerve could serve as a control for the axonal versus hematogenous source of the virus. Wild type mice were inoculated, sacrificed everyday and the eyes, optic nerves and brain were prepared for immunohistological examination. Cells in the intermediate layer were the first to be infected. Later, the virus spread to the layer of the ganglion cells. Viral antigens also appeared in the lateral geniculate nucleus and nowhere else in the thalamus which demonstrates axonal transport. Therefore, the source of infection of myelin and oligodendrocytes and astrocytes in the optic nerve is the virus which is transported in axons.

Their last hypothesis was the myelin is the portal of entry of the virus into the white matter oligodendrocytes. They used the shiverer mutant to distinguish between the two possibilities. That wither the virus traffics from the axon to myelin cytoplasmic channels and from there to the oligodendrocyte cell body. Or that the oligodendrocyte cell bodies could be infected by the virus diffusing from degenerating axons and the infection could spread outwardly from the cell body to the myelin. The only difference between the wild type and the mutant is that the amount of myelin is reduced. First they compared the level of virus replication in the retina of the wild type and shiverer mice. The minus strand viral RNA was found only in infected cells, so therefore they could say the assay was not biased by viral particles in the inoculum. Then they compared the time of arrival of the infection in the lateral geniculate nucleus for wild type and mutant mice using immunoflueorescence and found no difference. It can be concluded that the amount of virus transported in the optic nerve must be very similar in wild-type and shiverer mice.
They then scanned frozen sections of the optic nerves and found that 50-100% of infected cells were oligodendrocytes in wild type mice were in contrast no infected oligodendrocytes were found in the shiverer mutation.
Therefore the mutant considerably hindered the infection of oligodendrocytes by axonally transported virions.

The authors found in summary that the myelin is infected by axonally transported virus and that the infections spreads secondarily from the myelin to the oligodendrocyte cell body. This traffic is interrupted by the shiverer mutation. It can thus be deduced that infecting myelin cytoplasm is essential for the persistence of Theiler’s virus in the CNS.

Critique

I found this paper to be very interesting, their experiments were well documented and their results can be important for the medical community, because they warrant looking for a similar phenomenon in other persistent infections of the nervous system, including those that infect humans. While I enjoyed and understood the paper, it was very technical. If a reader did not have any knowledge of immunological techniques they would not understand what the authors did. They did not explain much of the principles behind the techniques in their paper. Most of their diagrams were also very technical and if the reader had no experience in these techniques it would be hard to gather any information from them. Though overall, the paper was very well done and generally easy to understand.

The Nervous System

The nervous system is by far the most complex system in the human body, formed by millions of nerve cells which are assisted by even more glial cells (Junqueira and Carneiro, 2005). Because of its complexity, yet how simple things seem to flow makes the nervous system, in my view, the moist interesting and definitely my favorite. This blog is going to focus mostly on the tissues of the central nervous system, but I will briefly mention the peripheral nervous system.

The nervous system is distributed throughout the body as a way of communication. The nervous system is divided into two major components, the central nervous system (CNS) and the peripheral nervous system (PNS) (Becker Kleinsmith and Hardin, 2002). The central nervous system is comprised of the brain and the spinal cord while the peripheral nervous system consists of nerve fibers and small aggregates of nerve cells outsisde of the CNS (Junqueira and Carneiro, 2005).
Figure 1-1. The general functional organization of the central and peripheral nervous sysems (Junqueira and Carneiro, 2005).

The peripheral nervous system can be subdivided into the somatic nervous system, which controls voluntary movements, and the autonomic nervous system, which controls involuntary activities of the cardiac muscles and of the smooth muscles in the gastrointestinal tract, blood vessels and a variety of secretory glands (Becker et al, 2002). The autonomic nervous system can be divided again into the sympathetic and the parasympathetic nervous systems these act antagonistically, the sympathetic nervous system prepares the body for stress, and the parasympathetic comes the body down after stress (Hill Wyse and Anderson, 2004).

Cells that make up the nervous system can be divided into neurons and glial cells (Becker et al, 2002). Neurons usually show numerous long processes and are responsible for the reception, transmission and processing of a stimuli. They trigger certain cell activities, and release neurotransmitters. Glial cells have short processes, they support and protect the neuron, they participate in the activity, nutrition and the defense of the central nervous system (Junqueira and Carneiro, 2005).

A typical neuron is composed of the dendrites, the cell body and the axon (Junqueira and Carneiro, 2005)

Figure 1-2 The structure of a Typical Motor Neuron (a) A diagram of a typical motor neuron. (b) A scanning electron micrograph of the cell body and dendrites of a motor neuron. (Becker et al, 2002).

The cell body is the part that contains the nucleus and the surrounding cytoplasm (Junqueira and Carneiro, 2005). It contains extensions or branches, called processes (Becker et al, 2002). There are two types of cell processes, the ones that receive signals and ones that conduct signals, called dendrites and axons respectively (Becker et al, 2002). The cell body of most neurons receives a number of nerve endings that pass on either excitatory or inhibitory stimuli that have been generated in other cells. They usually have a spherical and unusually large pale staining nucleus with a prominent nucleolus. When the appropriate stain is used, the rough ER and free ribosomes appear under the light microscope as basophilic granular areas called Nissel bodies, as seen in figure 1-4. The number of Nissel bodies varies depending on the neuronal type and functional state, being particularly abundant in large nerve cells like motor neurons. Neurofilaments are abundant in the cell body and when viewed under the light microscope and fixed in silver, they form neurofibrils (Junqueira and Carneiro, 2005).

The Dendrites receive the synapses and are the principle signal reception and processing sites of the neurons. Each neuron would have many dendrites which increase the receptive area of the cell, this allows for one neuron to receive many stimuli and integrate them from the surrounding nerve cells. Dendrites become thinner in diameter as the branch out away from the cell body (Junqueira and Carneiro, 2005).

The axon is a cylindrical process which varies in length and diameter that depends on the type of neuron. The axons orginate in a short pyrimad shaped region known as the axon hillock (Junqueira and Carneiro, 2005). The plasma membrane of the within the axon is called the axolemma and its contents the axoplasm (Becker et al, 2002). The initial segment is the part of the axon between the axon hillock and the start of the mylenation, at this point the excitatory and inhibitory stimulations are summed which results in the decision to pass on the stimulus or not (Junqueira and Carneiro, 2005).

Neurons and their processes are variable in their size and in their shape, they can be large enough to be visible to the naked eye, or like the cell bodies of the granule cells of the cerebellum be among the smallest cells in the body. Neurons can be placed into one of three categories multipolar, bipolar and pseudounipolar. Multipolar neurons, have more than two processes, one being the dendrites and the other being the axon. Bipolar neurons have one dendrite and one axon and pseudounipolar neurons have a single process that is close to the cell body and divides into two branches, it then forms a T shape with one branch extending to the peripheral ending and the other to the CNS (Junqueira and Carneiro, 2005).

Figure 1-3. Simplified view of the three main types of neurons, according to their morpholigical characteristics (Junqueira and Carneiro, 2005).

In the body, most of the neurons are multipolar, some examples of bipolar neurons are the ones found in the cochlear and vestibular ganglia also the ones in the retina and olfactory mucosa. The ones found in the cranial ganglia and in the spinal ganglia are the pseudounipolar neurons ( Junqueira and Carneiro, 2005).

Neurons can be subdivided into three basic types based on their functions: sensory neurons, motor neurons, and interneurons . Sensory neurons are a diverse group of cells that are specialized for detection of stimuli, they provide a stream of information to the brain about the body and its environment. For example, photoreceptors in the retina, olfactory neurons and various touch neurons located in the skin. Motor neurons transmit signals from the central nervous system to the muscles or glands that they stimulate. Interneurons process signals from other neurons and relay the information to other parts of the nervous system (Becker et al, 2002).

Figure 1-4 Photomicrograph of a motor neuron, a very large cell, from the spinal cord. The cytoplasm contains a great number of nissl bodies. The large cell process is a dendrite. Pararosaniline-toluidine blue (PT) stain. Medium magnification. (Junqueira and Carneiro, 2005).

Surrounding the axons of nerve cells are a layer of insulating cells known as the glial cells (Becker et al, 2002). Glial cells are also found in the mammalian brain, they surround the cell body and all their process that occupy the interneuronal spaces. There are five types of glial cells in the nervous system: Oligodendrocytes, Schwann cells, astrocytes, ependymal cells and Microglia. Oligodendrocytes produce the myelin sheath that surrounds the neurons and provides the electrical insulation of the central nervous system, see figures 1-5 and 1-6D. These cells can branch and serve more than one nerve cell and its process (Junqueira and Carneiro, 2005).

Schwann cells have the same function as the oligodendrocytes but they are located around in the axons of the peripheral nervous system. As opposed to the oligodendrocytes, only one schwann cell can wrap around the axon of only one neuron (Junqueira and Carneiro, 2005).

The astrocytes are star shaped cells that have multiple radiating process and they are the most numerous glial cells. The astrocytes function to bind the neurons to the capillaries and to the pia matter. There are two main types of astrocytes called fibrous astrocytes and protoplasmic astrocytes. Fibrous astrocytes are located in the white matter, they have a few long processes, they can be seen in figure 1-5 and figure 1-6A. Protoplasmic astrocytes are located in the gray matter and many short processes, they can be seen in figure 1-5 and figure 1-6B. Astrocytes also control the ionic and chemical environment of the neurons, they influence neuronal survival and activity throughout their ability to regulate the extracellular environment (Junqueira and Carneiro, 2005).

Ependymal cells are columnar cells lining the ventricles of the brain and central canal of the spinal cord (Junqueira and Carneiro, 2005).

Microglia are small cells that have short irregular process, they are derived from precursor cells in the bone marrow. They are involved with inflammation and repair of adult cranial nerves of the central nervous system, they secrete a number of immunoregulatory cytokines and they dispose of unwanted cellular debris caused by CNS legions (Junqueira and Carneiro,2005). They can be seen figure 1-5 and figure 1-6C.

Figure 1-5 Drawings of neuroglial cells as seen in slides stained by metallic impregnation. Note that only astrocytes exhibit vascular end-feet, which cover the walls of blood capillaries.

Figure 1-6 Photomicrographs (prepared with Golgi stain) of glial cells from the cerebral cortes A: Fibrous astrocytes, showing blood vessls B:Protoplasmic astrocyte showing brain surface C: Microglial cell D: Oligodendrocytes

Function of the Neuron

The function of a nerve cell is to pass on an electrical impulse. A neuron at rest has a membrane potential of -60mV, this is set by a specific balance of ion gradients and ion permeabilities which is largely controlled by voltage gated sodium and potassium channels, in a resting cell these are usually closed. An electrical stimulus causes the sodium channels to open which makes the membrane ten times more permeable to sodium than to potassium, this causes the sodium to rush into the cell making it positive on the inside instead of negative, the membrane potential peaks at 40 mV. This is called depolarization, once triggered it propagates down the axon toward the axon terminal. Once the membrane potential has peaked, it quickly repolarizes, this happens because the sodium channels are inactivated and remain closed preventing any sodium from entering the cell, and also the potassium channels open so potassium can leave the cell. This eventually restores the membrane potential to the resting -60mV. The wave of depolarization propagates down the axon till it reaches the axon terminal where it causes the terminal buds to release a neurotransmitter which diffuses into the synaptic gap, the space between one neurons axons and another neurons dendrites, and then either causes an inhibitory or excitatory response in the postsynaptic neuron that influences whether that neuron will go through depolarization. The glial cells aid in the propagation of the wave of depolarization because when they wrap around axons they leave a space between each myelin sheath known as nodes of nodes of Ranvier. Myelination decreases the ability of the neuronal membrane to retain electrical charge which permits a depolarization event to spread further and faster than it would along a nonmyelinated axon. This is important in times where a very fast response is beneficial (Becker et al, 2002).

The Central Nervous System

The Central nervous system consists of cerebrum, cerebellum and the spinal cord, it has little connective tissue and is a soft gel-like organ (Junqueira and Carneiro, 2005).
When stained, they show regions of white matter and regions of grey matter, the white is composed of myelinated axons and the myelin producing oligodendrocytes (Junqueira and Carneiro, 2005). The grey matter contains neuronal cell bodies, dendrites and the unmyelinated portions of the axons and glial cells. The grey matter is prevalent at the surface of the cerebrum and the cerebellum, as compared to the white matter which is present in more central regions (Junqueira and Carneiro, 2005).

In the cerebral cortex, the grey matter has six layers of cells of all different forms and sizes. There are both sensory neurons and motor neurons in the different regions of the cerebral cortex which control voluntary movements. The cells of the cerebral cortex have functions related to the integration of sensory information and the initiation of voluntary motor responses, see figure 1-7(Junqueira and Carneiro, 2005).

Figure 1-8 Silver-stained section of cerebral cortex showing many pyrimad-shaped neurons with their processes and a few glial cells. Medium magnification (Junqueira and Carneiro, 2005)

There are three layers of the cerebellum: a molecular layer, a Purkinje layer and a granular layer.The molecular layer is the outermost layer, it cells are less dense than those in the granular layer, it also has the dendrites of the Purkinje cells that occupy the Purkinje layer. The Purkinje layer composed of large Purkinje cells, these have a conspicuous cell body and have highly developed dendrites. The Granular layer is the inner layer, it is formed by small neurons which are very compacted together (Junqueira and Carneiro, 2005). The cerebellum and its layers can be seen in figures 1-8 and 1-9.

Figure 1-8 Photomicrograph of the cerebellum. The staining procedure used (H&E) does not reveal the unusually large dendritic aborization of the Purkinje cell. Low magnification (Junqueira and Carneiro, 2005).

Figure 1-9 Section of the cerebellum with dintinct Purkinje cells. H&E stain. Medium magnification. (Junqueira and Carneiro, 2005).

The Spinal cord has white matter on the outside and grey matter on the inside usually forming an H shape and has large and multipolar neurons The horizontal bar in the H of the grey matter forms the central canal, the legs of the H form the anterior horns and the arms of the H form the anterior horns.The central canal is remnat of the luman of the embryonic neural tube and the anterior horns have the motor neurons whose axons make up the ventral roots of spinal nerves, the anterior horns receive sensory fibers from neurons in the spinal ganglia (Junqueira and Carneiro, 2005). A cross section of the spinal cord can be seen in figure 1-10.

Figure 1-10 Cross Section of the spinal cord in the transition between grey matter (Below) and white matter (Above). PT Stain. Medium Magnification (Junqueira and Carneiro, 2005)

The skull and the vertebral column protect the central nervous system, but they are aided by a membrane of connective tissue called meninges. The meninges are the dura matter, arachnoid and pia matter. Dura matter is the external layer and is composed of dense connective tissue with the periosteum of the skull. The arachnoid has two compartments: a layer that connects with the dura matter and a layer of trabeculae connecting to the pia matter. The Pia matter is loose connective tissue that contains the blood vessels (Junqueira and Carneiro).

Pathology

There are many different diseases of the nervous system, including Parkinson’s, tumors of the nervous system, Alzheimer’s and multiple sclerosis, to only name a few.
Multiple sclerosis, also known as MS, is the most common cause of neurologic disability, it is a autoimmune disease, which happens when the body mistakes its own self cells as antigens and attacks them. In the case of MS the body attacks the central nervous system. It is usually diagnosed between the ages of 20 and 40 and some symptoms are mild including numbness in the limbs or very severe like paralysis or loss of vision. People who have the disease produce autoreactive T lymphocytes that participate in the formation of inflammatory lesions along the myelin sheath of nerve fibers. The cerebrospinal fluid of patients with active MS contain activated T lymphocytes that infiltrate the brain and cause the characteristic inflammatory lesions which destroys the myelin. This breakdown in the myelin sheath leads to numerous neurological problems (Kindt Goldsby and Osborne, 2007).
Like most autoimmune diseases, the cause of MS is not well known, though some studies have proposed a link between MS and infection by certain viruses. It is known that some viruses can cause demyelinating diseases so it is easy to speculate that a viral infection plays a role in the development of MS (Kindt et al, 2007).

References

Becker Wayne M. , Kleinsmith Lewis J. , and Hardin, Jeff. The World of the Cell fitfh edition. Benjimen Cummings. San Francisco, CA. 2002.

Hill Richard W. , Wyse Gordon A. , and Anderson Margaret. Animal Physiology. Sinauer Associates, Inc. Massachusetts USA. 2004.

Junqueira Luiz Carlos, and Carneiro Jose. Basic Histology text and atlas eleventh edition. McGraw-Hill. USA. 2005.

Kindt Thomas J. , Goldsby Richard A. , and Osborne Barbara A. Kuby Immunology sixth edition. W.H Freeman and Company. New York. 2007.