AXONAL DEGENERATION

Axonal degeneration inevitably follows the death of the neuron of which it is a part. A severed or severely damaged axon undergoes distal degeneration without usually provoking death of the proximal part of the neuron, although this does undergo a series of structural and metabolic changes (i.e. axon reaction and chromatolysis, see below). Within days, the distal part of the axon fragments and the surrounding myelin sheath breaks up into ovoids. Over the next 3 weeks or so, the axon and myelin debris are taken up by macrophages, which infiltrate the degenerating fiber tracts. Several methods can be used to demonstrate degenerating nerve fibers in the CNS. These include specialized silver impregnation techniques and stains for degenerating myelin and for lipid (Fig. 1.1).

Marchi’s method, and oil red O or other stains for neutral lipid are particularly useful. During the first 2–3 weeks after injury when most of the products of fiber degeneration are still extracellular, their demonstration by Marchi’s method requires staining of unembedded tissue that has not fixed long in formalin. If there has been a longer period of time since the injury and much of the debris has been taken up by macrophages, Marchi’s method can be used on frozen sections and the staining is not affected by the duration of formalin fixation. Degeneration of fiber tracts can be demonstrated for several months with stains for neutral lipid, and for several years by Marchi’s method. In addition, immunohistochemistry for macrophage markers (e.g. with antibodies to CD68 and HLA class 2 antigen) is useful for detection of degenerating fiber tracts.

AXON REACTION AND CHROMATOLYSIS

Damage to the axon provokes a series of morphologic and biochemical changes in the neuronal cell body. These are collectively referred to as the axon reaction (Figs 1.2, 1.3). The changes include disruption and dispersion of Nissl bodies (chromatolysis) associated with rearrangement of the cytoskeleton and marked accumulation of intermediate filaments. The axon reaction is conspicuous in large neurons with axons that project into the peripheral nervous system and in some of the larger neurons with central projections. Chromatolysis is not visible on conventional light microscopy of small neurons or certain large neurons such as the cerebellar Purkinje cells, but changes can be demonstrated in these cells by electron microscopy and immunohistochemistry.

SWOLLEN NEURONS

Swollen or ballooned neurons are a feature both of the axon reaction and of a variety of diseases in which perikaryal changes occur independently of axonal damage (Table 1.2). Histologically, they appear as distended, weakly-staining cells with large, relatively clear nuclei (Fig. 1.4). Occasionally the cells contain small vacuoles. In conventionally processed tissues an artefactual lacuna is often present around the abnormal cell. Swollen or ballooned neurons can be demonstrated with several immunohistochemical markers (Fig. 1.5, Table 1.3).

TRANS-SYNAPTIC NEURONAL DEGENERATION AND OLIVARY HYPERTROPHY

Neurons within some nuclei in the CNS atrophy and degenerate in response to deafferentation. Examples are neurons in the lateral geniculate nucleus, which degenerate after optic nerve or tract lesions, and neurons in the pontine nuclei, which degenerate after interruption of descending frontopontine afferent fibers. Neurons in the inferior and accessory olivary nuclei undergo an unusual form of trans-synaptic degeneration after a destructive lesion (such as an infarct) of the ipsilateral central tegmental tract (Fig. 1.6). The olivary ribbon as a whole becomes thickened and neurons show marked enlargement, cytoplasmic vacuolation, and some dispersion of Nissl bodies. Olivary hypertrophy is associated with the development of palatal myoclonus in some patients.

HYPOXIC CELL CHANGE

Neurons are especially vulnerable to damage from hypoxia, which causes the following distinctive histologic changes (Fig. 1.7):

Histologic changes identical to these can be induced in neurons by hypoglycemia or by exposure to excessive amounts of excitotoxic neurotransmitters.

Cell stress proteins are expressed at an early stage of hypoxic injury and are demonstrable by immunohistochemical techniques.

Neurons in certain parts of the brain that are especially vulnerable to hypoxic damage are:

The pattern of regional susceptibility to hypoxia differs between infants and adults (see Chapters 2 and 8).

FERRUGINATION

Although dead neurons usually undergo liquefaction or are removed by phagocytosis, they may instead become encrusted and replaced by mineral salts, a process termed ferrugination. This is particularly prominent in the infant brain in response to hypoxic–ischemic damage (Fig. 1.8).

MARINESCO BODIES

These are small spherical nuclear inclusions that are brightly eosinophilic and are often seen in neurons of the adult substantia nigra (Fig. 1.9a). They may also occur in other neurons, such as the pyramidal cells of the hippocampus and neurons in the tegmentum of the brain stem. Marinesco bodies are most common in the elderly and in dementia with Lewy bodies. They have an increased prevalence in neurons containing Lewy bodies.

Ultrastructurally, Marinesco bodies are composed of filaments with the same diameter as intermediate filaments, and may be derived from the nuclear lamins. They are immunoreactive for ubiquitin, an 8 kD polypeptide involved in the degradation of many abnormal or short-lived proteins (Fig. 1.9b).

VIRAL INCLUSIONS

Nuclear inclusions are a feature of infection by several viruses (see Chapters 13 and 14), for example cytomegalovirus infection, which produces particularly striking nuclear inclusions (Fig. 1.10).

Hirano bodies

These are brightly eosinophilic rod-shaped or elliptical cytoplasmic inclusions that may appear to overlap the edge of a neuron (Fig. 1.11). They are immunoreactive for actin, actin-associated proteins, and caspase-cleaved TDP43.

Ultrastructurally, Hirano bodies consist of a regular lattice of multiple layers of parallel 10–12 nm filaments, the filaments in one layer being transversely or diagonally oriented with respect to those in the adjacent layers.

Hirano bodies are most numerous in the CA1 field of the hippocampus. Their density in the stratum lacunosum increases until middle-age and declines gradually thereafter, except in chronic alcoholics, in whom the density may continue to increase. In the elderly, the number of Hirano bodies increases in the stratum pyramidale, and they are particularly numerous in this region in Alzheimer disease, Pick’s disease, some sub-types of Creutzfeldt–Jakob disease, and Guam parkinsonism–dementia.

EOSINOPHILIC THALAMIC NEURONAL INCLUSIONS

These are smaller than Hirano bodies, but are otherwise similar in shape and tinctorial staining characteristics. They occur in thalamic neurons in normal middle-aged and elderly individuals, but are commoner in patients with myotonic dystrophy.

ROD-LIKE CYTOPLASMIC INCLUSIONS

These eosinophilic cytoplasmic inclusions occur in large neurons of the caudate nucleus. The inclusions resemble loose bundles of twigs and, like the thalamic neuronal inclusions described above, are common in patients with myotonic dystrophy. However, they are sometimes an incidental finding in elderly patients who do not have neurologic or muscular disease. The rod-like inclusions stain strongly with phosphotungstic acid/hematoxylin (Fig. 1.12).

OTHER FILAMENTOUS NEURONAL INCLUSIONS

There are several other types of neuronal inclusion that comprise or include elements of the cytoskeleton and usually occur in the context of specific neurodegenerative diseases (Table 1.4). These inclusions are described in more detail and illustrated in the sections concerned with the relevant diseases.

LAFORA BODIES

These are composed of polyglucosans (polymers of sulfated polysaccharides) and are similar to corpora amylacea in composition and staining characteristics (see below). They are present in large numbers in Lafora’s disease (see Chapter 7), both in the CNS and in certain peripheral tissues such as sweat glands, liver, and skeletal muscle. Lafora body formation has been linked to aberrant glycogen hyperphosphorylation. The inclusions usually have a round core that is intensely periodic acid–Schiff (PAS)-positive (Fig. 1.13). Spicules of the core may radiate outwards, into a surrounding zone of less intensely PAS-positive material.

EOSINOPHILIC INCLUSIONS IN THE INFERIOR OLIVES

A characteristic feature of aging is the development of ill-defined eosinophilic inclusions in the neurons of the inferior olivary nuclei. These inclusions are intensely immunoreactive with antibodies to ubiquitin (Fig. 1.14) and should not be confused with lipofuscin, which also accumulates with age (see below) and is particularly abundant in neurons of the inferior olivary nuclei.

COLLOID INCLUSIONS

Colloid inclusions are round eosinophilic inclusions that usually occur in neurons in the hypoglossal nuclei (Fig. 1.15), but may be seen in other large neurons, particularly in the elderly. Electron microscopy shows that these inclusions result from dilatation of the endoplasmic reticulum by amorphous material. The importance of recognizing colloid inclusions, which do not have any clinical significance, is that they are occasionally confused with inclusions that are clinically significant such as Lewy bodies, pale bodies, and hyaline inclusions of motor neuron disease (see Chapter 27).

BUNINA BODIES

These are small beaded eosinophilic inclusions seen in motor neurons in motor neuron disease. Cystatin C, transferrin, and peripherin have been detected in Bunina bodies. Ultrastructurally, they appear as electron-dense membrane-bound bodies (see Chapter 27).

INCLUSIONS DERIVED FROM THE ACID VESICLE SYSTEM

The acid vesicle system consists of endosomes, lysosomes, and lysosome-derived dense bodies.

Lipofuscin (Fig. 1.16) is produced by oxidation of lipids and lipoproteins within the lysosomal system. It appears as orange-brown granular material in sections stained with hematoxylin and eosin, and is acid-fast (as demonstrated by the long Ziehl–Neelsen method) and autofluorescent under ultraviolet light. The granules are also sudanophilic and stain with PAS and Schmorl’s stain. Lipofuscin accumulates with aging in neurons and glia, particularly in:

The lipofuscin accumulation is increased in some neurodegenerative disorders such as Alzheimer disease and motor neuron disease.

Granulovacuolar degeneration. This term describes the accumulation of vacuoles containing small round dense bodies (granulovacuoles) (Fig. 1.17). The dense bodies are ubiquitinated and react with antibodies to some epitopes of the microtubule-associated tau protein. The immunohistochemical data have been interpreted as suggesting that dense bodies are derived from partial degradation of tau protein within lysosomes. Although anti-phosphoTDP43 antibody is sensitive for detecting granulovacuolar degeneration, TDP43 proteinopathies are not associated with an increase in granulovacuoles. Granulovacuolar degeneration is seen in normal aging after the sixth decade, predominantly in the hippocampal formation. Neurons in the CA1 field are most severely affected and, in descending order of severity, those in the prosubiculum, CA2, CA3, and CA4 fields. The density of hippocampal neurons showing granulovacuolar degeneration is increased in patients with Alzheimer disease and Pick’s disease, in whom granulovacuolar degeneration may also occur in neurons in the subcortical nuclei.

Storage products in neurometabolic diseases. The accumulation of material within the acid vesicle system is a feature of many neurometabolic diseases including the lysosomal storage disorders, as well as certain other metabolic disorders caused by non-lysosomal enzyme defects. These disorders are considered in Chapter 23.

AXONAL SWELLINGS

Spheroids are axonal swellings. They are composed of neurofilaments, organelles, and other material that is normally conveyed along the axon by anterograde transport systems, but accumulates focally when these are impaired (Fig. 1.18a,b). Spheroids are a feature of axonal damage by diverse extrinsic insults, especially trauma (see Chapter 11) and infarcts. There may be numerous axonal spheroids around the edge of an infarct.

The term ‘torpedo’ is applied to Purkinje cell axonal swellings, which are a feature of a wide range of metabolic and degenerative cerebellar diseases. These swellings appear as fusiform eosinophilic structures in the cerebellar granule cell layer (Fig. 1.18c,d). They are well demonstrated by silver impregnation (see chapter 29).

The axonal swellings that develop when axonal transport is disrupted by neuronal metabolic dysfunction are usually termed dystrophic (Fig. 1.18e). These occur in certain nutritional deficiencies (particularly vitamin E deficiency, see Chapter 21) and inherited metabolic diseases (e.g. Niemann–Pick disease type C). Dystrophic axonal swellings are the principal pathologic abnormality seen in a group of conditions termed the neuroaxonal dystrophies (see Chapter 33).

The term ‘dystrophic neurite’ is also used to describe neuronal processes within the gray matter that are distended by tau protein or other abnormal ubiquitinated material. These occur in several neurodegenerative diseases (Table 1.5).

Dystrophic axons that react strongly with antibodies to ubiquitin and p62 are a feature of aging in the CNS, especially in the gracile and cuneate nuclei, the substantia nigra, the globus pallidus, and the anterior horns of the spinal cord.

In brains from people over 60 years of age, antibodies to ubiquitin label smaller dot-like structures in the neuropil of the cerebral cortex and in the white matter. These correspond to membrane-bound dense bodies in dystrophic neurites and foci of granular degeneration in myelin sheaths (Fig. 1.19).

SWELLING OF ASTROCYTES

This is a relatively rapid response to a wide range of stimuli. Acutely reactive astrocytes are swollen by hyaline eosinophilic cytoplasm and have enlarged vesicular nuclei (Fig. 1.21).

FIBRILLARY GLIOSIS

With time, reactive astrocytes proliferate and insinuate long cytoplasmic processes into the adjacent brain parenchyma. These processes contain bundles of glial intermediate filaments, which appear as fibrils in appropriately stained preparations; hence the term fibrillary gliosis. The abundance of glial intermediate filaments is readily demonstrated by immunostaining for GFAP. Two patterns of fibrillary gliosis are recognized:

ROSENTHAL FIBERS

Rosenthal fibers are inclusions that develop in astrocytes in chronic reactive and neoplastic proliferations. They are brightly eosinophilic structures of variable size and shape (Fig. 1.24a,b). On electron microscopy they contain admixed amorphous granular material and 10 nm diameter filaments (Fig. 1.24c). Immunohistochemistry reveals GFAP, αB-crystallin, 27 kD heat-shock protein, and ubiquitin at the periphery of the Rosenthal fibers (Fig. 1.24d–f), while the central hyaline region is generally unlabeled.

Rosenthal fibers are seen in longstanding reactive gliosis, especially when it occurs in the spinal cord, cerebellum, or hypothalamus. They are also a feature of pilocytic astrocytomas. Rosenthal fibers are abundant in Alexander’s disease and in giant axonal neuropathy.

GRANULAR BODIES

Granular bodies are found in regions of chronic astrocytic gliosis, usually in conjunction with Rosenthal fibers. They are clusters of round eosinophilic granules within the cytoplasm of astrocytes (Fig. 1.25) and express GFAP as well as αB-crystallin. Ultrastructural examination reveals membrane-bound dense bodies.

TAU-IMMUNOREACTIVE GLIA

Tau protein has long been known to be the main constituent of neurofibrillary tangles, which are neuronal inclusions in Alzheimer disease and several other disorders (see Chapters 28, 29, and 31), but the recognition that glial cells can accumulate tau protein is a relatively recent finding. Several different types of tau-immunoreactive inclusions have been described (Fig. 1.26, Table 1.6). See also Pathologic reactions of oligodendroglia, p.16.

CORPORA AMYLACEA

Corpora amylacea are spherical inclusions, predominantly in astrocyte processes (Fig. 1.27a,b), although they occasionally occur within axons. They range from 10 μm to 50 μm in diameter, consist largely of polyglucosans, and stain with hematoxylin, PAS, and methyl violet. Minor constituents include ubiquitin, heat-shock proteins, tau protein, complement proteins, and some oligodendrocyte proteins (i.e. myelin basic protein, proteolipid protein, galactocerebroside, and myelin oligodendrocyte glycoprotein). Ultrastructurally, corpora amylacea consist of densely packed 6–7 nm filaments that may be admixed with amorphous granular material and are not membrane bound.

Corpora amylacea increase in number with normal aging, particularly in subpial (Fig. 1.27c) and subependymal regions, around subcortical blood vessels, and in the spinal white matter. Their increase in number is greater in conditions where there has been atrophy and gliosis, including Alzheimer disease and other neurodegenerative disorders. Transglutaminase 1-mediated cross-linking and polymerization of cytoskeletal or cytoskeletal-associated proteins has been postulated as a mechanism of corpora amylacea formation.

The functions of corpora amylacea, if any, are not known. Suggestions include a role in the accumulation of inorganic materials from the blood and cerebrospinal fluid or in shielding immunogenic products of neuronal and oligodendroglial degeneration from lymphocytic recognition and autoimmune activation.

NUCLEAR CHANGES IN ASTROCYTES

Alzheimer type II astrocytes are seen in cortical and subcortical gray matter regions in liver failure. They have an enlarged vesicular nucleus with marginated chromatin, scanty cytoplasm, and little or no demonstrable GFAP (Figs 1.28–1.32) (see also Chapter 22).

DEMYELINATION AND REMYELINATION

Demyelination, or loss of normal myelin with relative preservation of axons, may result from selective destruction of the myelin sheath, or may be secondary to damage to the oligodendrocyte cell body. Demyelination is often immunologically mediated, as in multiple sclerosis (see Chapter 19) and acute disseminated encephalomyelitis (see Chapter 20), but can also be caused by toxic and metabolic insults (see Chapters 22 and 25), viral infection (see Chapter 13), compression (see Chapter 20), and ischemia (see Chapter 9). Areas of demyelination show loss of staining with Luxol fast blue, or solochrome cyanin (Fig. 1.33). Electron microscopy demonstrates the absence of myelin sheaths. Remyelinated nerve fibers have myelin sheaths that are abnormally thin in relation to the caliber of the axon (Fig. 1.33). Groups of remyelinated fibers are visible histologically as discrete areas of white matter in which staining of myelin is present but reduced (Fig. 1.33).

DYSMYELINATION

This term is sometimes used to describe the failure to form and/or maintain myelin sheaths that occurs in the leukodystrophies, a heterogeneous collection of genetically-determined disorders of white matter. Most of these dysmyelinating disorders are due to defects of peroxisomal or lysosomal metabolism. The leukodystrophies are considered in Chapters 5 and 23.

INTRAMYELINIC VACUOLATION

Several disorders of the CNS cause a distinctive pattern of edema in which the initial accumulation of fluid is in ‘vacuoles’ formed by separation of the layers of the myelin sheath along intraperiod lines. On light microscopy, this intramyelinic vacuolation gives the white matter a spongy appearance (Fig. 1.34). The causes include some mitochondrial encephalopathies (particularly Kearns–Sayre syndrome; see Chapter 24), Canavan’s disease and several disorders of amino acid metabolism (see Chapter 5), vitamin B12 deficiency (see Chapter 21), various toxins (see Chapter 25), and retroviral infections – vacuolar myelopathy due to HIV, and myelopathy due to HTLV-1 (see Chapter 13).

AXONAL DEGENERATION

As noted above, axonal degeneration, of whatever etiology, leads to fragmentation and degeneration of myelin, and its subsequent phagocytosis and degradation by macrophages. The degradation products can be detected by Marchi’s method, and oil red O or other stains for neutral lipid (Fig. 1.1). The infiltrating macrophages are well demonstrated with specific immunohistochemical markers (e.g. antibody to CD68).

INCLUSIONS

Oligodendrocytes accumulate cytoplasmic aggregates of filamentous or microtubular material in several neurodegenerative diseases. Inclusions that consist largely of tau protein are a feature of progressive supranuclear palsy, corticobasal degeneration, and frontotemporal lobar degeneration linked to chromosome 17 (FTDP-17). In these diseases (discussed in detail in Chapters 28 and 31), tau inclusions occur in the perinuclear oligodendrocyte cytoplasm where they often partially encircle the nucleus and are called ‘coiled bodies’ (Fig. 1.35). In progressive supranuclear palsy, delicate strands of aggregated tau also occur in the in the inner and outer loops of the myelin sheath, forming inclusions know as ‘glial threads’ or ‘interfascicular threads’. In multiple system atrophy (see Chapter 28), oligodendrocytes form ‘glial cytoplasmic inclusions’ (GCIs) – curved, filamentous, flame- or sickle-shaped inclusions that often partly encircle the nucleus (Fig. 1.35). They are readily demonstrable by Gallyas silver impregnation, and immunohistochemically with antibodies to α-synuclein (Fig. 1.35). They can also be labeled with antibodies to ubiquitin, and some antibodies to tau. Electron microscopy shows GCIs to consist of microtubular structures with a coating of granular material.

ATROPHY

There is loss of cytoplasm in ependymal cells, which become flattened. Nuclei lose their basal position and encroach upon the rest of the cytoplasm in affected ependymal cells (Fig. 1.38). Atrophy usually occurs in association with hydrocephalus or cerebral atrophy (e.g. in encephalopathies of childhood, age-related dementias).

DISCONTINUITY

Stretching and tearing of ependyma may occur with ventricular enlargement, as in hydrocephalus. Discontinuity may also be a sequel of severe atrophy.

SUBVENTRICULAR GLIOSIS (GRANULAR EPENDYMITIS, EPENDYMAL GRANULATIONS)

This probably occurs within 1–2 weeks of ependymal tearing or other injury. Proliferation of astrocytes occurs in the subventricular region, and astrocytic processes produce a dense meshwork that extends along the denuded ventricular lining and may form nodular protrusions into the ventricular cavity (Fig. 1.39). The formation of these subventricular glial nodules is sometimes termed ‘granular ependymitis’, although it is not often part of an inflammatory process. When subventricular gliosis is associated with ventricular hemorrhage, hemosiderin-laden macrophages are present.

SUBEPENDYMAL ROSETTES AND TUBULES

These are tiny collections of ependymal cells, forming small cord-like, tubular or rosette-like structures, which are situated below an ependymal surface. The angles of the lateral ventricles and the cerebral aqueduct are sites of predilection. Subependymal rosettes and tubules are occasionally incorporated within subventricular glial nodules.

INFLAMMATION AND INFECTION

True ependymitis may result from ependymal trauma, irritation by toxins (e.g. vincristine), ventricular hemorrhage, or intraventricular inflammation (e.g. in association with purulent ventriculitis). Several viruses also show tropism for ependyma, including mumps (a possible cause of acquired atresia of the aqueduct) and several members of the Herpes virus family (e.g. cytomegalovirus and varicella-zoster virus).

BIONDI BODIES

These are intracellular accumulations of amyloid fibrils in ependymal cells and choroid plexus epithelium. The number of Biondi bodies increases with age, and they are consistently present in large numbers in the elderly. In choroid plexus epithelium the fibrils may be arranged in wispy strands or large cytoplasmic rings, which are also known as Biondi rings (Fig. 1.40). In ependymal cells the fibrils appear as irregular strands rather than rings. The fibrils consist of tightly packed bundles of straight 10 nm filaments, which stain with thioflavin S and less intensely with Sirius red or Congo red. They have been reported to react with antibody to Aβ-amyloid.

Biondi bodies persist even after the autolysis of other tissues and their density in homogenates of choroid plexus has been used to provide a rapid indication of the age of an unidentified corpse.

 Resident microglia, which undergo little turnover with hematogenous monocytes and occur within the CNS parenchyma, though they may abut blood vessels.

 Perivascular microglia, which occur within the perivascular basal lamina and undergo turnover with hematogenous monocytes.

 Increased entry of hematogenous monocytes into the CNS (Fig. 1.41c).

 Proliferation of resident microglia.

 Expression or secretion of a range of proteins, most of which are concerned with antigen presentation and inflammation – these include major histocompatibility (MHC) class I and II antigens and several cytokines.