Neuropathology and muscle biopsy techniques

Introduction

In neuropathology, as in most other areas of histological practice, hematoxylin and eosin (H&E) remains the most useful and commonly used stain, as it demonstrates most cell types well with good detail. As with other histological disciplines, neuropathology relies more on immunohistochemistry and cytogenetics and less on the more capricious and obscure preparations of the past. Nonetheless, a number of reliable and useful tinctorial and metal-based stains remain in common usage.

Neuropathological practice increasingly stresses the importance of molecular and cytogenetic aspects of disease ( ). This is most clearly illustrated by the ‘layered’ approach to diagnosis in the recent WHO tumor classification ( ) whereby morphological, immunohistochemical and cytogenetic data are built up into a final ‘integrated’ diagnosis. Such genetic techniques include in situ hybridization, Sanger and pyrosequencing and ‘next generation’ sequencing ( ). Currently, many of these are performed in supra-regional cytogenetics laboratories but, as these methods increase in number and importance, it is essential that histology staff become conversant with them.

Muscle biopsy techniques are included in this chapter as these methods tend to be performed in the areas of the laboratory designated for neuropathology.

The components of the normal central nervous system

The nervous system can be subdivided into the central, peripheral and autonomic nervous systems. This chapter will concern itself principally with the central nervous system and secondarily with the peripheral nervous system and muscle. The principal components of the central nervous system are:

• Neurons

• Glial cells

• Meninges

• Blood vessels.

The neuron is an excitable cell which is responsible for processing and transmitting information. Neurons communicate with each other via intercellular interfaces called synapses. At the synapse, an electrical impulse in the presynaptic neuron causes it to release a chemical transmitter which diffuses across a narrow gap to influence the electrical activity of the postsynaptic target neuron. Neurons have several components ( Fig. 18.1 ):

• The cell body (or ‘soma’), containing various subcellular organelles responsible for the metabolic upkeep of the cell.

• The nucleus, which resides in the cell body and is the site of storage of the cell’s genetic code in DNA.

• The dendritic tree, a complex of cell processes responsible for receiving synaptic inputs from other neurons.

• The axon, an elongated fiber which transmits electrical impulses away from the soma to synapses with either other neurons or muscle fibers. This may be a meter or more in length in the case of the lower motor neurons which reside in the lower (lumbar) spinal cord and innervate the muscles of the lower leg.

Two naturally occurring pigments may be observed to accumulate in the brain with age, lipofuscin and neuromelanin. Both are believed to represent cellular waste products. Lipofuscin is a yellow-brown, autofluorescent, granular substance composed of peroxidized protein and lipids. It is seen in larger neurons, such as the lower motor neurons of the spinal cord and pyramidal cells of the hippocampus in Alzheimer pathology. Large amounts of lipofuscin-like pigment also accumulates in inherited neuronal ceroid lipofuscinoses, of which Batten’s disease is the most common ( ). Neuromelanin is most commonly seen in the cytoplasm of neurons of the substantia nigra and the locus ceruleus. It is the cause of the macroscopic pigmentation of these structures. Under the microscope, it is a dark brown, granular material which is believed to be the by-product of oxidative metabolism of catecholamines ( ).

Glial cells are the support cells of the nervous system. They are diverse in nature, and the principal types and their functions follow.

• Astrocytes have a number of functions. Firstly, they maintain the extracellular ion and neurotransmitter balance. Secondly, they are involved in repair and scarring responses to brain damage and finally astrocytes form part of the blood–brain barrier, protecting the brain from harmful blood-borne substances.

• Oligodendroglia form myelin, a phospholipid sheath around nerve axons which enhances the speed of conduction of impulses.

• Ependymal cells line the ventricles of the brain and central canal of the spinal cord.

• Microglia are the native immune cell of the nervous system of monocyte/macrophage lineage.

Neuropil is a term used to denote the feltwork of neuronal processes in which neuron cell bodies reside. Central nervous system tissue is classically subdivided into gray matter, containing the majority of neuronal cell bodies and little myelin, and white matter, which is predominantly formed of myelinated axons and few neurons.

The meninges form three layers of protective covering over the brain. The outer layer, beneath the skull, is the dura mater; a tough, fibrous membrane. The arachnoid mater is a more delicate, fibrillary covering which lies inside the dura mater and is more closely adherent to the brain surface, but it does not invaginate into the surface infoldings of the brain (or sulci). The pia mater is the most delicate covering. It is closely apposed to the brain surface, following its contours down into the depths of sulci.

Techniques for staining neurons

Tinctorial stains for Nissl substance

Hematoxylin and eosin (H&E) preparations demonstrate the most important features of neurons. However, Nissl preparations are also popular for examining the basic architecture of neural tissue and its components. These are often combined with the luxol fast blue myelin stain. Granules of Nissl substance are found in the cell body ( Fig. 18.1 ) and correspond to rough endoplasmic reticulum. They are basophilic due to the associated nucleic acid ( ). Many basic dyes e.g. neutral red, methylene blue, azure, pyronin, thionin, toluidine blue and cresyl fast violet stain Nissl substance. Variation in the stain used, pH and degree of differentiation allow preparations to label either Nissl substance alone, or Nissl substance in combination with cell nuclei.

Motor neurons generally have coarse (‘tigroid’) Nissl substance, and regions such as the anterior horns of the spinal cord, where these cells are abundant, are good tissues to use when learning these stains ( Fig. 18.2 ). For paraffin wax-embedded sections of formalin-fixed tissue, the cresyl fast violet stain is reliable and relatively straightforward. As such it is by far the most commonly used Nissl preparation. Toluidine blue may also be used, whilst Einarson’s gallocyanin method, being more suited to alcohol-fixed tissue, is largely unused ( ).

Cresyl fast violet (Nissl) stain for paraffin wax sections

Fixation

Alcohol, Carnoy’s or formalin.

Sections

Paraffin wax, 7–10 μm or 25 μm (see Note b).

Preparation of stain

Cresyl fast violet	0.5 g
Distilled water	100 ml

Differentiation solution

Glacial acetic acid	250 μl
Alcohol	100 ml

Method

• 1.

  Dewax sections and bring to water.

• 2.

  Cover with filtered cresyl fast violet, stain for 10–20 minutes.

• 3.

  Rinse briefly in distilled water.

• 4.

  Differentiate in 0.25% acetic alcohol until most of the stain has been removed (4–8 seconds).

• 5.

  Briefly pass through absolute alcohol into xylene and check microscopically.

• 6.

  Repeat steps 4 and 5 if necessary, giving less differentiation when repeating.

• 7.

  Rinse well in xylene and mount in DPX.

Results

Nissl substance	purple/dark blue
Neurons	pale purple/blue
Cell nuclei	purple-blue

Notes

• a.

  If only Nissl substance is required to be demonstrated, the stain is acidified with 0.25% acetic acid.

• b.

  Estimation of cortical neuronal density is made on 25 μm thick sections.

• c.

  The cresyl violet method can be used as a counterstain when demonstrating myelin with the Kluver and Barrera method (page 312).

Myelin

Myelin forms an electrically insulating sheath around axons. It is approximately 80% lipid and 20% protein and is formed from sheet-like processes of glial cells concentrically wrapped multiple times around the axon. This greatly improves the speed and efficiency of impulse conduction along the axon. Myelin is formed by oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. A single oligodendrocyte may myelinate multiple axons in its vicinity, whereas a single Schwann cell only myelinates a single segment of a single axon. Loss of myelin, as is seen in multiple sclerosis in the central nervous system or in Guillain-Barré syndrome in the peripheral nervous system, can be severely debilitating.

Modern tinctorial stains for myelin are simple and reliable and can be performed on formalin-fixed paraffin wax-processed tissue. Many can be combined with a Nissl stain to demonstrate neuronal localization. We also find that the myelin staining is more intense and satisfactory when combined with Nissl staining than without. Older methods may give more even and consistent staining, but are considerably more time consuming and have fallen from use ( ). Luxol fast blue, solochrome cyanine and IHC preparations are now favored. Many antisera are used as markers of myelination, the most useful being myelin basic protein, proteolipid protein and myelin associated glycoprotein ( ). Given the reliability and simplicity of the tinctorial stains, IHC myelin markers are rarely used in routine diagnostic neuropathology and remain the preserve of research laboratories. Immunohistochemistry for S-100 is useful in the diagnosis of tumors derived from Schwann cells both in the central nervous system and peripherally ( ).

Luxol fast blue is a copper phthalocyanine dye which is employed in myelin staining of paraffin wax-processed tissue ( ). This can be combined with cresyl violet or hematoxylin to outline cellular architecture ( Fig. 18.4 ) or with periodic acid-Schiff (PAS) to demonstrate myelin degradation products in demyelinating disease in central nervous system tissue only.

Myelin loss may occur in a region of brain damaged by any of a number of processes, e.g. trauma, neoplasia, multiple sclerosis or toxic insult. It may also occur secondary to the loss of axons emanating from any damaged brain region. In modern practice, degeneration of myelinated tracts is most commonly demonstrated by showing loss of normal myelin staining by either luxol fast blue or solochrome cyanine preparations, or by showing a microglial reaction using CD68 IHC ( ).

The neuroglia

This term refers to the supporting cells of the central nervous system and comprises ependymal cells, astrocytes, oligodendrocytes and microglia. As is becoming a recurrent theme, IHC is increasingly replacing tinctorial stains for their identification.

Astrocytes

These cells have multiple, fine processes and in their reactive state are ‘star-shaped’, hence the name. On standard H&E sections, only the nucleus of resting astrocytes is distinct, as the cell body cannot be discerned from background neuropil. These nuclei are slightly larger with more open granular chromatin than those of the more compact oligodendrocyte. Modern neuropathology relies most heavily on GFAP ( Fig. 18.5 ) IHC for the demonstration of astrocytes, although antibodies to S-100, αB-crystallin and glutamine synthetase may also be used.

Astrocytes are principally classified into protoplasmic and fibrous forms. These are similar in function, but protoplasmic astrocytes have shorter, thicker, highly branched processes and are generally found in the gray matter; fibrous astrocytes have longer, thinner, less-branched processes and usually reside in the white matter. Astrocytic reactions in the cerebellum are characterized by Bergmann or radial astroglia which have processes running radially from the Purkinje cell layer of the cortex to the pial surface.

In response to injury of the brain parenchyma, astrocytes react by increasing in size with a more prominent, eosinophilic cytoplasm. The nucleus moves from a central to a more eccentric position within the cell cytoplasm and processes become more prominent. Astrocytic gliosis is a response to permanent injury, whereby astrocytes proliferate to fill tissue defects with a fibrous glial scar.

In neuro-oncology, astrocytic differentiation is best demonstrated by GFAP IHC. GFAP immunoreactivity is also seen in other tumors including ependymoma, oligodendroglial tumors and choroid plexus tumors ( ). Astrocytic tumors also label with vimentin and S-100, but these are also seen in many other tumor types, rendering them of little use for differential diagnoses. Astrocytes occasionally show cross-reactivity as seen for the pan cytokeratin AE1/AE3 ( ); it is expedient to use other cytokeratin markers such as CAM5.2 or MNF116 to exclude the diagnosis of epithelial tumors.

The proliferation marker Ki-67 (MIB-1) is often used as an aid to grading tumors and to help differentiate reactive from neoplastic astrocytic populations. The latter will tend to have a higher number of nuclei labeled with this marker.

A recently introduced tool for the diagnosis of diffuse oligodendroglial and astrocytic neoplasms is the use of antibodies to isocitrate dehydrogenase 1 (IDH1) carrying the R132H mutation. Approximately 74% of mid-grade (WHO grade II and III) diffuse astrocytomas and oligodendrogliomas have mutations of either IDH1 or IDH2 . Tumours with IDH mutations have a better prognosis than those without. The R132H mutation in IDH1 accounts for 90% of such mutations ( ). Commercially available antisera to this mutant protein may be used to differentiate astrocytomas and oligodendrogliomas from their mimics as well as to provide some prognostic prediction ( ).

Neurodegeneration

Neurodegenerative conditions are largely diseases of old age. As the population ages, these conditions place an increasing burden on health and social care systems. This, together with the escalation in research into neurodegeneration in recent years, has resulted in an increasing workload on neuropathology units.

It is often the case that a definitive diagnosis of any neurodegenerative condition cannot be made until autopsy. In most studies, the accuracy of clinical diagnosis of the cause of a dementing illness is in the order of 75%. A neuropathological autopsy does not benefit the deceased, but does have wider benefits, namely:

• It can yield data and tissue to assist research.

• It provides epidemiological data, allowing the prevalence of different neurodegenerative diseases to be monitored.

• Autopsy findings are often of considerable educational benefit for both senior and junior clinicians as well as pathologists.

• An increasing number of neurodegenerative conditions are familial, often with known causative mutations. Accurate neuropathological characterization can therefore guide genetic counseling.

The pathological characterization of neurodegenerative disease is a staged process. The first step is removal of the brain, sometimes with the spinal cord. Following formalin fixation, the brain is examined macroscopically both intact and after slicing (usually coronally). Appropriate blocks of tissue are taken, processed and paraffin wax embedded for microscopy.

In the majority of cases, neuropathological assessment of neurodegeneration tends to broadly focus on dementing illnesses and motor degeneration, with considerable overlap between the two. Investi-gations of dementia commonly uncover one, or a combination of, three types of pathology, namely: Alzheimer’s disease, vascular dementia or dementia with Lewy bodies. A small number of dementia cases show frontotemporal lobar degeneration (FTLD). The neurodegenerative diseases of the motor system which are diagnosed at autopsy tend to focus on motor neuron disease (also known as amyotrophic lateral sclerosis) and conditions which cause Parkinsonian clinical features. Other neurodegenerative diseases of the motor system e.g. Huntington’s disease, spinocerebellar ataxia and Friedreich’s ataxia tend to be diagnosed by genetic tests.

The microscopic examination of the brain for neurodegeneration is often an iterative process, whereby an initial examination is performed using fairly standard preparations, most favoring H&E. After this, more specialist preparations, usually IHC, are performed ( ).

Many neurodegenerative diseases are characterized by accumulations (or inclusions) of protein, and for the majority there are now commercially available antisera. These diseases are often classified by the particular protein which forms the pathological aggregates characterizing the disorders and these inclusion bodies are detailed in Table 18.1 . These categories are:

• The tauopathies , e.g. Alzheimer’s disease, Pick’s disease, supranuclear palsy, corticobasal degeneration and argyrophilic grain disease.

• The synucleinopathies , Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy.

• Prion disorders and TDP-43 proteinopathies , motor neuron disease, frontotemporal lobar degeneration with TDP-43.

Ubiquitin is a small regulatory protein which binds misfolded or other aberrant proteins, and labels them for destruction by the proteasome. Many dementing illnesses are characterized by accumulations of ubiquitylated protein; the location and form of these accumulations can provide valuable diagnostic clues. Therefore, IHC for ubiquitylated proteins is often a useful early step in neuropathological diagnosis and we favor antibodies to p62. This protein binds ubiquitylated proteins and shuttles them to the proteasome ( ). Antibodies to p62 can be used to label pathological, ubiquitylated aggregates of tau, α-synuclein and TDP-43 ( ). Further, p62 IHC has greater specificity for pathological aggregates than ubiquitin IHC, leaving non-pathological features unlabeled.

As noted above, autopsies in neurodegeneration principally concern dementia and motor system degenerative diseases, many of which overlap. Of the dementing illnesses, the vast majority are diagnosed as Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTLD. However, given the considerable public health concerns surrounding prion diseases, cases will also occasionally be assessed for this diagnosis.

Alzheimer’s disease is characterized by ubiquitylated accumulations of tau and β-amyloid. Hyperphosphorylated tau forms flame-shaped neuronal intracytoplasmic inclusions known as neurofibrillary tangles, and neuritic fibrillary deposits known as neuropil threads. β-Amyloid is formed from aggregates of peptides generated by the cleavage of amyloid precursor protein (a membrane-associated protein of unknown function) by β- and γ-secretases. Deposits of β-amyloid become surrounded by dilated and distorted neuronal processes to form senile plaques. Senile plaques and neurofibrillary tangles are the histological hallmarks of Alzheimer’s disease ( Figs. 18.6 and 18.9 ).

Vascular dementia is an umbrella term for a variety of conditions characterized either by multiple large or small regions of infarction throughout the brain, or by smaller numbers of infarcts in functionally important structures such as the thalamus or hippocampus ( ). The picture is somewhat complicated by the fact that many cases with a heavy burden of vascular pathology will additionally have a coexistent burden of Alzheimer or Lewy body-type pathology.

Dementia with Lewy bodies is characterized by neocortical and limbic neuronal cytoplasmic inclusions of α-synuclein i.e. the Lewy bodies which give this condition the name. Lewy bodies were first described in Parkinson’s disease, where they are visible as round, intensely eosinophilic, hyaline intracytoplasmic neuronal inclusions in the substantia nigra and locus ceruleus of the midbrain and brainstem. They are composed of ubiquitylated α-synuclein and can therefore be detected by IHC for antisera to these proteins or p62 ( ).

Frontotemporal lobar degeneration (FTLD) is an umbrella term for a number of neuropathological entities, all of which predominantly manifest clinically as frontotemporal dementia. The different forms of FTLD are classified by the immunoreactivity of their characteristic proteinaceous intracytoplasmic inclusions ( ). However, this is a complex and constantly evolving field and classifications tend to rapidly become outdated in their finer details. In essence, the majority of cases are classified as tauopathies due to their accumulations of intracytoplasmic tau. Of the non-tauopathies, most cases show intracytoplasmic inclusions of ubiquitylated, phosphorylated TDP-43 (designated FTLD-TDP), and a few are characterized by inclusions of FUS/TLS or neurofilament. A small residuum is characterized by intracytoplasmic inclusions with immunoreactivity to p62 and ubiquitin alone (FTLD-UPS), or no observable reactivity at all, so-called ‘dementia lacking distinctive histology’.

The prion diseases are rare neurodegenerative disorders. They are of considerable interest due to the inherited nature of some forms; the tragic and rapid progression of these conditions; the public health monitoring necessary because of their transmissible nature, and similarly the risk to laboratory staff.

The normal function of prion protein is unclear, and yet it is a normal constituent of cell membranes and most highly expressed in neurons. When misfolded, it is capable of causing disease. In humans, most cases are sporadic, largely Creutzfeldt-Jakob disease (CJD); approximately 10% of cases are familial, and a few have been caused by medical intervention. Prion disease due to the consumption of matter containing misfolded prion proteins is implicated in variant CJD (vCJD) and (historically) kuru ( ).

Clinically, sporadic CJD is characterized by a rapidly progressive multifocal neurological dysfunction, sleep disturbance, myoclonic jerks, ataxia and a terminal severe globalized cognitive impairment. Death occurs after approximately 8 months ( ).

Pathologically, CJD is characterized by neuronal loss, gliosis, spongiform change (giving them their alternative name of spongiform encephalopathies), and the deposition of protease-resistant prion protein. From a practical perspective, it should be noted that prion protein is a normal constituent of the nervous system. The pathological form is detected by first treating sections with a protease enzyme to eradicate immunoreactivity to normally folded, physiological prion protein. This leaves immunoreactivity only for pathological, misfolded, protease-resistant prion protein.

Parkinsonism is characterized by tremor, hypokinesia, rigidity and postural instability. It is most commonly caused by Parkinson’s disease, which is pathologically characterized by Lewy bodies in the brainstem and midbrain structures (see above and Fig. 18.7 ). Less common causes include progressive supranuclear palsy and corticobasal degeneration, which are both characterized by neuronal intracytoplasmic accumulations of hyperphosphorylated tau, and multiple system atrophy, which is characterized by glial cytoplasmic inclusions of α-synuclein.

Most cases of motor neuron disease , in common with FTLD-TDP, are characterized by neuronal and glial cytoplasmic accumulations of hyperphosphorylated, ubiquitylated TDP-43 ( Fig. 18.8 ).

Following consideration of the principal forms of dementia as described above, it will be apparent that in order to provide a full diagnostic service, a laboratory should have access to optimized IHC for hyperphosphorylated tau, β-amyloid, α-synuclein, p62 or ubiquitin (ideally the former), TDP-43, neurofilament and protease-resistant prion protein.