based on selective expression of Cre recombinase. Wnt1Cre, which marks probable risk factor for MCC because it is prevalent on sun-exposed
all neural crest cells, failed to label Merkel cells; however, Krt14Cre, a skin and in immunocompromised patients. Intriguingly, MCC has been
marker of keratinocyte-derived cells, labelled Merkel cells in all skin linked to a newly described virus, Merkel cell polyomavirus (Feng et al
areas (Morrison et al 2009, Van Keymeulen et al 2009). Moreover, 2008), raising the possibility that viral infection plays a role in MCC
Merkel cells are replenished from epidermal progenitors in mature skin pathogenesis. Active areas of investigation include identifying a causal
(Van Keymeulen et al 2009, Woo et al 2010, Doucet et al 2013). Finally, link between MCC and polyoma viral infection, and defining mecha-
Merkel cells were abolished by conditional deletion of a developmental nisms that underlie the aggressiveness of MCC.
transcription factor, Atoh1, driven by Krt14Cre but not Wnt1Cre (Morrison
Acknowledgements
et al 2009, Van Keymeulen et al 2009). Together, these studies provide
a starting point to define transcriptional networks that bestow a neuro- Thanks to members of the Lumpkin laboratory for helpful comments
sensory fate in epidermal cells (Bardot et al 2013, Lesko et al 2013). and Ms Blair Jenkins for assistance with the figure. The author is sup-
Despite remarkable progress in Merkel-cell biology, important ques- ported by the National Institutes of Health (R01 AR051219 and R01
tions about human Merkel cells remain unanswered. Do Merkel cells NS073119).
REFERENCES
Bardot ES, Valdes VJ, Zhang J et al 2013 Polycomb subunits Ezh1 and Ezh2 Lesko MH, Driskell RR, Kretzschmar K et al 2013 Sox2 modulates the func-
regulate the Merkel cell differentiation program in skin stem cells. tion of two distinct cell lineages in mouse skin. Dev Biol 382:15–26.


EMBO J 32:1990–2000. Maksimovic S, Nakatani M, Baba Y et al 2014 Epidermal Merkel cells are
Bhatia S, Afanasiev O, Nghiem P 2011 Immunobiology of Merkel cell carci- mechanosensory cells that tune mammalian touch receptors. Nature
noma: implications for immunotherapy of a polyomavirus-associated 509:617–21.
cancer. Curr Oncol Rep 13:488–97. Maricich SM, Morrison KM, Mathes EL et al 2012 Rodents rely on Merkel
Doucet YS, Woo SH, Ruiz ME et al 2013 The touch dome defines an epider- cells for texture discrimination tasks. J Neurosci 32:3296–300.
mal niche specialized for mechanosensory signaling. Cell Rep 3: Maricich SM, Wellnitz SA, Nelson AM et al 2009 Merkel cells are essential
1759–65. for light-touch responses. Science 324:1580–2.
Feng H, Shuda M, Chang Y et al 2008 Clonal integration of a polyomavirus Merkel F 1875 Tastzellen und Tastkörperchen bei den Hausthieren und beim
in human Merkel cell carcinoma. Science 319:1096–100. Menschen. Archiv f mikrosk Anat 11:636–52.
Haeberle H, Fujiwara M, Chuang J et al 2004 Molecular profiling reveals Moll R, Moll I, Franke WW 1984 Identification of Merkel cells in human skin
synaptic release machinery in Merkel cells. Proc Natl Acad Sci U S A by specific cytokeratin antibodies: changes of cell density and distribu-
101:14503–8. tion in fetal and adult plantar epidermis. Differentiation 28:136–54.
Halata Z, Grim M, Bauman KI 2003 Friedrich Sigmund Merkel and his Morrison KM, Miesegaes GR, Lumpkin EA et al 2009 Mammalian Merkel
‘Merkel cell’, morphology, development, and physiology: review and cells are descended from the epidermal lineage. Dev Biol 336:76–83.
new results. Anat Rec 271A:225–39.
Van Keymeulen A, Mascre G, Youseff KK et al 2009 Epidermal progenitors


Harms PW, Patel RM, Verhaegen ME et al 2013 Distinct gene expression give rise to Merkel cells during embryonic development and adult
profiles of viral- and nonviral-associated Merkel cell carcinoma revealed homeostasis. J Cell Biol 187:91–100.
by transcriptome analysis. J Invest Dermatol 133:936–45.
Woo SH, Ranade S, Weyer AD et al 2014 Piezo2 is required for Merkel-cell
Iggo A, Muir AR 1969 The structure and function of a slowly adapting touch mechanotransduction. Nature 509:622–6.
corpuscle in hairy skin. J Physiol 200:763–96.
Woo SH, Stumpfova M, Jensen UB et al 2010 Identification of epidermal
Ikeda R, Cha M, Ling J et al 2014 Merkel cells transduce and encode tactile progenitors for the Merkel cell lineage. Development 137:3965–71.
stimuli to drive Aβ-afferent impulses. Cell 157:664–75.
Yamashita Y, Ogawa H 1991 Slowly adapting cutaneous mechanoreceptor
Johnson KO 2001 The roles and functions of cutaneous mechanoreceptors. afferent units associated with Merkel cells in frogs and effects of direct
Curr Opin Neurobiol 11:455–61. currents. Somatosens Motor Res 8:87–95.


e19
4.1
YRATNEMMOC
COMMENTARY
1.4
Metaplasia
Jonathan MW Slack, Leonard P Griffiths, David Tosh
Metaplasia is the appearance, in adult life, of a patch of tissue that examples of structures in which a number of different tissue types arise
normally belongs elsewhere in the body (Slack 1985, Slack 2007). from a simple epithelial tube in the embryo.
Generally, it is understood that the metaplasia originated in situ and The clinical importance of metaplasias stems from the fact that they
has not migrated from elsewhere. Therefore, metastatic tumour depos- often predispose to cancer. For example, carcinomas of the bronchus
its, for example, are not considered to be metaplasias, and neither are usually arise within areas of squamous metaplasia (Auerbach et al
tissues misplaced during embryonic development, such as thyroid resi- 1961) and carcinomas of the stomach usually arise in regions of intes-
dues in the thyroglossal duct. tinal metaplasia (Busuttil and Boussioutas 2009).
Metaplasias are very diverse (Willis 1962). They include ectopic bone Metaplasias arise because the combination of gene activities that
and cartilage arising from connective tissue, squamous metaplasia of defines the tissue type in embryonic development becomes altered in
glandular epithelia, and substitution of one glandular tissue for another. adult life. The genes in question encode a limited number of key tran-
The latter group is of particular interest from a biological point of view. scription factors. For example, the transcription factor p63 is essential
These metaplastic transformations are normally between tissue types for the formation of squamous epithelia (Koster et al 2004) and is
that originated in embryonic development as neighbours in a common always found to be expressed in patches of squamous metaplasia. In
cell sheet. They may remain neighbours into adult life or may become experiments with mice, ectopic expression of the Cdx2 gene, normally
separated. For example, intestinal metaplasia of the stomach is a well- necessary for intestinal development, leads to patches of intestinal tissue


studied type of metaplasia in which patches of small intestine-like in the stomach (Silberg et al 2002, Mutoh et al 2002). Conversely, dele-
epithelium are found in the stomach (Stemmermann and Hayashi tion of Cdx2 from the intestine leads to oesophagus-like or stomach-like
1968, Gutierrez-Gonzalez and Wright 2008). Intestinal metaplasia patches in the intestine (Gao et al 2009, Stringer et al 2012). In tissues
found in the urinary bladder contains cell types and markers normally that are maintained by continuous renewal from a population of stem
found in the colon (Sung et al 2006). The bladder is not part of a cells, it is the stem cells whose character becomes switched in an initiat-
common cell sheet with the intestine in the adult, but in the embryo it ing event. Once the change has occurred, it is irreversible.
is, originating from the allantoic diverticulum of the hindgut. Glandular A well-studied example of metaplasia is Barrett’s metaplasia,
metaplasias are particularly common in the alimentary canal and in the commonly known as Barrett’s oesophagus (Falk 2002, Gilbert et al
female reproductive tract (Kurita 2011), perhaps because these are both 2011) (Fig. 1.4.1). This is characterized by the presence of columnar
Z-line
Z-line
BM BM
SSQE
A B C SSQE
SSQE
SSQE
BM BM
D E
Fig. 1.4.1 Barrett’s metaplasia. A, Normal endoscopic appearance showing the junction between gastric columnar and oesophageal squamous
epithelium (Z-line). B–C, Endoscopies showing two cases of Barrett’s oesophagus. The Z-line has moved proximally and is very irregular. There are
islands of squamous tissue within the columnar region. D, Histological view, haematoxylin and eosin. E, Histological view, immunostained for CDX2
(brown). Scale bar 200 microns. Abbreviations: BM, metaplastic epithelium (Barrett’s metaplasia); SSQE, stratified squamous epithelium.


METAplAsiA
e20
1
NOiTCEs
epithelium in the distal oesophagus, of which some resembles intesti- epithelium, the tissue also contains mucous glands connected by ducts
nal epithelium possessing goblet cells. Barrett’s metaplasia is important to the lumen, which are located in the submucosa but originate from
because it is a risk factor for the development of oesophageal adeno- the original embryonic oesophageal epithelium. Barrett’s metaplasia
carcinoma, a cancer whose incidence rose considerably in the late twen- probably arises by transformation of the basal cells of the squamous
tieth century (Hvid-Jensen et al 2011). In less developed countries, most epithelium, or from the mucous glands, or both (Nicholson et al 2012,
oesophageal cancers are squamous cell carcinomas, arising from the Leedham et al 2008).
normal squamous lining of the oesophagus but, in the Western world, The predisposing cause of Barrett’s metaplasia is gastro-oesophageal
adenocarcinomas are now more common. reflux, in which stomach acid, and bile from the intestine, enter the
At least some metaplasias are monoclonal, i.e. a particular focus oesophagus (Souza 2010). It is known that this environment can induce
consists of tissue all derived from a single cell. This is the case for intes- in oesophageal cells expression of some of the key genes involved
tinal metaplasia of the stomach, where neighbouring metaplastic crypts in intestinal development, such as those encoding CDX factors and
arise by fission (Gutierrez-Gonzalez et al 2011). In Barrett’s metaplasia, hepatocyte nuclear factors (Eda et al 2003, Debruyne et al 2006, Piessen
patches of crypts can also be monoclonal (Nicholson 2012), although et al 2007). Obesity is a well-known risk factor for reflux, partly because
the lesion as a whole is polyclonal. A key property of metaplastic foci of effects on intra-abdominal pressure and probably also through the


is that the new tissue is in a situation of competitive growth with the effects of cytokines released from fat (Corley et al 2007). Barrett’s meta-
old tissue that surrounds it. Unless the metaplastic tissue can outgrow plasia may progress to dysplasia and to adenocarcinoma. Progression
the surrounding tissue, then the transformation of a single stem cell is associated with loss of the tumour suppressor gene CDKN2A, encod-
would not give rise to a macroscopic lesion. However, this aspect of ing the protein p16, which is an inhibitor of cell division; and TP53,
epithelial biology remains poorly understood. encoding the protein p53, required for death of abnormal cells
The exact cell of origin of Barrett’s metaplasia is still not known (Spechler et al 2010, Chen et al 2011). Studies of the clonal structure
(Barbera and Fitzgerald 2010). The columnar lined epithelium of Bar- of Barrett’s metaplasia have shown that is it complex, containing
rett’s metaplasia is in continuation with the columnar lined gastric cell populations derived from several original progenitors, with
mucosa and appears as a proximal displacement of the junction with various combinations of abnormalities arising from somatic mutation
the stratified squamous epithelium (the Z-line). However, it is generally (Leedham et al 2008).
thought not to be a phenomenon of cell migration alone, as animal Gastro-oesophageal reflux is normally treated with proton pump
experiments indicate that it is possible to elicit patches of metaplasia inhibitors that reduce the levels of stomach acid. This improves associ-
separated from the gastric epithelium (Gillen et al 1988, Goldstein ated oesophagitis but does not usually result in regression of any Bar-
et al 1997). The normal oesophagus originates from an endodermal rett’s metaplasia. Barrett’s metaplasia as such is not normally treated
tube lined with columnar epithelium, which becomes squamous but, if it progresses to dysplasia, it may be extirpated by a variety of
between about 6–7 weeks of gestation. Although lined with squamous endoscopic techniques (Lim and Fitzgerald 2013).
REFERENCES


Auerbach O, Stout AP, Hammond EC et al 1961 Changes in bronchial epi- Kurita T 2011 Normal and abnormal epithelial differentiation in the female
thelium in relation to cigarette smoking and cancer of the lung. N Engl reproductive tract. Differentiation 82:117–26.
J Med 265:253–67.
Leedham SJ, Preston SL, McDonald SA et al 2008 Individual crypt genetic
Barbera M, Fitzgerald RC 2010 Cellular origin of Barrett’s metaplasia and heterogeneity and the origin of metaplastic glandular epithelium in
oesophageal stem cells. Biochem Soc Trans 38:370–3. human Barrett’s oesophagus. Gut 57:1041–8.
Busuttil RA, Boussioutas A 2009 Intestinal metaplasia: a premalignant Lim YC, Fitzgerald RC 2013 Diagnosis and treatment of Barrett’s oesopha-
lesion involved in gastric carcinogenesis. J Gastroenterol Hepatol 24: gus. Br Med Bull 107:117–32.
193–201.
Mutoh H, Hakamata Y, Sato K et al 2002 Conversion of gastric mucosa
Chen H, Fang Y, Tevebaugh W et al 2011 Molecular mechanisms of Barrett’s to intestinal metaplasia in Cdx2-expressing transgenic mice. Biochem
esophagus. Dig Dis Sci 56:3405–20. Biophys Res Commun 294:470–9.
Corley DA, Kubo A, Levin TR et al 2007 Abdominal obesity and body mass Nicholson AM, Graham TA, Simpson A et al 2012 Barrett’s metaplasia
index as risk factors for Barrett’s esophagus. Gastroenterology 133: glands are clonal, contain multiple stem cells and share a common
34–41. squamous progenitor. Gut 61:1380–9.
Debruyne PR, Witek M, Gong L et al 2006 Bile acids induce ectopic expres- Piessen G, Jonckheere N, Vincent A et al 2007 Regulation of the human
sion of intestinal guanylyl cyclase C through nuclear factor-kappa B and mucin MUC4 by taurodeoxycholic and taurochenodeoxycholic bile


Cdx2 in human esophageal cells. Gastroenterology 130:1191–206. acids in oesophageal cancer cells is mediated by hepatocyte nuclear
Eda A, Osawa H, Satoh K et al 2003 Aberrant expression of CDX2 in Barrett’s factor 1 alpha. Biochem J 402:81–91.
epithelium and inflammatory esophageal mucosa. J Gastroenterol 38: Silberg DG, Sullivan J, Kang E et al 2002 Cdx2 ectopic expression induced
14–22. gastric intestinal metaplasia in transgenic mice. Gastroenterology 122:
Falk GW 2002 Barrett’s esophagus. Gastroenterology 122:1569–91. 689–96.
Gao N, White P, Kaestner KH 2009 Establishment of intestinal identity and Slack JMW 1985 Homeotic transformations in man: implications for the
epithelial-mesenchymal signaling by Cdx2. Dev Cell 16:588–99. mechanism of embryonic development and for the organization of
epithelia. J Theor Biol 114:463–90.
Gilbert EW, Luna RA, Harrison VL et al 2011 Barrett’s esophagus: a review
of the literature. J Gastrointest Surg 15:708–18. Slack JMW 2007 Metaplasia and transdifferentiation: from pure biology to
the clinic. Nat Rev Molec Cell Biol 8:369–78.
Gillen P, Keeling P, Byrne PJ et al 1988 Experimental columnar metaplasia
in the canine oesophagus. Br J Surg 75:113–15. Souza RF 2010 The role of acid and bile reflux in oesophagitis and Barrett’s
metaplasia. Biochem Soc Trans 38:348–52.
Goldstein SR, Yang GY, Curtis SK et al 1997 Development of esophageal
metaplasia and adenocarcinoma in a rat surgical model without the use Spechler SJ, Fitzgerald RC, Prasad GA et al 2010 History, molecular mecha-
of a carcinogen. Carcinogenesis 18:2265–70. nisms, and endoscopic treatment of Barrett’s esophagus. Gastroenterol-
ogy 138:854–69.


Gutierrez-Gonzalez L, Graham TA, Rodriguez-Justo M et al 2011 The clonal
origins of dysplasia from intestinal metaplasia in the human stomach. Stemmermann GN, Hayashi T 1968 Intestinal metaplasia of the gastric
Gastroenterology 140:1251–60.e6. mucosa. A gross and microscopic study of its distribution in various
disease states. J Natl Cancer Inst 41:627–34.
Gutierrez-Gonzalez L, Wright NA 2008 Biology of intestinal metaplasia in
2008: more than a simple phenotypic alteration. Dig Liver Dis 40: Stringer EJ, Duluc I, Saandi T et al 2012 Cdx2 determines the fate of post-
510–22. natal intestinal endoderm. Development 139:465–74.
Hvid-Jensen F, Pedersen L, Drewes AM et al 2011 Incidence of adenocarci- Sung MT, Lopez-Beltran A, Eble JN et al 2006 Divergent pathway of intesti-
noma among patients with Barrett’s esophagus. N Engl J Med 365: nal metaplasia and cystitis glandularis of the urinary bladder. Mod
1375–83. Pathol 19:1395–401.
Koster MI, Kim S, Mills AA et al 2004 p63 is the molecular switch for initia- Willis RA 1962 The Borderland of Embryology and Pathology. London:
tion of an epithelial stratification program. Genes Dev 18:126–31. Butterworths.


e21
5.1
YRATNEMMOC
COMMENTARY
Electron microscopy in the
1.5
twenty-first century
Roland A Fleck
Introduction
Biological electron microscopy (EM) is pivotal in life science research.
A measure of its ubiquity is the diversity of its capacity, e.g. three-
dimensional structural information about proteins and viruses at Å
resolution; three-dimensional reconstructions of cellular organelles and
tissues (from a few tens of nanometres to hundreds of micrometres);
and two-dimensional and topographic information and elemental
microanalysis at subcellular resolution. The capacity of EM is now
further enhanced by correlative light and electron microscopy (CLEM)
workflows, where light microscopy (LM) events are co-localized with
underlying ultrastructure. In its simplest form, LM generates a ‘Google
Earth’-like map to identify areas of interest, which are then studied at
the ultrastructural level by EM, so overcoming both the physical size
limitations imposed on samples by EM and the diffraction limits of
resolution imposed by LM (Williams and Carter 2009). CLEM can also
be combined with advanced EM sample preparation and imaging tech-
niques to provide previously unheralded levels of cytoarchitectural
insight into biological systems (McDonnald 2009, Plitzko et al 2009,
Sartori et al 2007).
However, biological EM is not without its challenges. The electron
Fig. 1.5.1 A Masson trichrome stain of a renal biopsy taken for diagnostic
beam is generated under vacuum at pressures and temperatures that are histopathology. The arrow highlights a glomerular capillary, fringed with
nominally incompatible with liquid water, yet water is the most abun- foot processes, which would be selected for investigation at higher
dant cellular constituent. Moreover, carbon-based life forms also have resolution by transmission electron microscopy (see Video 1.5.1). For
poor contrast in the electron microscope because they are composed diagnostic pathology, the whole glomerulus would be examined. Key: red,
mainly of light elements. In fact, carbon is so ‘transparent’ in the elec- keratin; blue/green, collagen; light red/pink, cytoplasm. (Image courtesy of
tron microscope that it is often employed as a film to support biological M Balys.)


samples. To overcome these caveats, hydrated ‘live’ tissue is converted
to a ‘fixed’ state; conventional protocols employ a series of steps, includ-
ing chemical fixation, alcohol dehydration and resin infiltration. Heavy
metal salts are added for positive staining of fixed and embedded speci-
mens or negative staining of whole structures that have been deposited translate to, and to advance, clinical research and diagnostic pathology
on a support film (Glauert and Lewis 1998). More recently, cryo-fixation (Fig. 1.5.1, Video 1.5.1).
has been adopted to allow the ‘solidification of a biological specimen
by cooling with the aim of minimal displacement of its components’ Limitations of traditional processing
(Steinbrecht and Zierold 1987). By using low temperature as a physical techniques and current protocols
fixation strategy, the morphology and dimensions of the living material
are retained and soluble cellular components are not displaced, which
Traditional EM processing strategies introduce artefacts (perturbations
means that processing artefacts commonly encountered in more con-
to structure) (Fig. 1.5.2) that affect tissue structure, e.g. by shrinkage or
ventional room-temperature EM techniques are either reduced or
swelling of the tissue under examination, the shrinkage of cellular
removed. Cryo-EM often allows direct observation of specimens that
organelles, and/or the extraction or redistribution of cellular constitu-
have not been stained or chemically fixed.
ents such as lipids, proteins and DNA (Boyde and Maconnachie 1979).
Most chemical fixatives react with proteins and cross-link peptide
EM in pathology
chains as part of their action (Glauert and Lewis 1998). These reactions
can be highly deleterious to epitopes, significantly compromising
Despite the decline in diagnostic histopathology by EM from the height immunohistochemical studies.
of its popularity in the 1980s, EM remains an important diagnostic tool Cryo-fixation has two distinct advantages over chemical fixation
in several well-defined areas. Indeed, guidance issued by the UK’s Royal (Nermut and Frank 1971, Nermut 1991). It is rapid (measured in milli-
College of Pathologists cites and supports the Association of Clinical seconds), which means that the sample is preserved, hydrated and in a


Pathologists Best Practice No. 160, which states that ‘Many respondents ‘close-to-life’ state at the point of initiating fixation, and ensures simul-
[to a review of laboratory practice in renal pathology] expressed the taneous immobilization of all macromolecular components (Robards
opinion that to carry out evaluation of renal biopsy specimens without and Sleytr 1995). Many protein networks are labile and prone to disrup-
at least having the availability of electron microscopy is negligent’ (de tion with even a slight osmotic or temperature change; cryo-fixation
Haro and Furness 2012). Other specialized areas include the diagnosis minimizes these deleterious effects. Cryo-techniques allow the study of
of primary ciliary dyskinesia; certain skin/connective tissue disorders, biological samples with improved ultrastructural preservation and may
e.g. inherited bullous lesions and Ehlers–Danlos syndrome; and spe- facilitate the study of dynamic processes. Rapid freezing prevents arte-
cialized areas of ophthalmic pathology (de Haro and Furness 2012). factual aggregations of proteins because tissue fixation and processing
Diagnostic transmission electron microscopy (TEM) in the clinical can be performed with no or minimal use of cross-linking fixatives,
setting has been largely stagnant and there has been little change in which means that samples retain higher antigenicity (Kellenberger
procedures or application for decades. In marked contrast, TEM in the 1991, Hayat 2000, Claeys et al 2004).
research setting has experienced an explosion in capacity and capability, This level of preservation relies on vitrification (the transformation
and there is considerable opportunity for twenty-first century EM to of water from a liquid to an amorphous state without inducing the


ElECTRON MiCROsCOpY iN ThE TwENTY-fiRsT CENTuRY
e22
1
NOiTCEs
Room temperature Room temperature specimen
specimen handling handling and processing
Plunge freezing
Warm/ ‘live sample’ Physically fixed vitrified specimen
High-pressure freezing
Fixation
Freeze fracture VTF
Chemical fixation
Cryo-ultramicrotomy
Tokuyasu CEMOVIS
Freeze-substitution Freeze fracture
Processing
Ultramicrotomy
Replica
Conventional room
Tokuyasu/
temperature sample
immunolabelling technique
preparation techniques
Visualization
Imaging by either SEM or TEM at conventional imaging Imaging by either cryo-SEM or cryo-TEM
conditions of temperature and pressure under cryogenic conditions
Fig. 1.5.2 An overview of electron microscopy specimen preparation workflow. Areas to the left of the dashed line are at room temperature, indicating
that samples can be handled without the requirement for specialist cryo-operating procedures. Samples to the right of the dashed line must be handled
in a manner suitable for vitreous biological samples, taking specific care to pre-cool tools before approaching or manipulating samples. Arrows denote
the direction of workflow and key stages required to process a sample from ‘living/close to life’ state to final image. Vitreous thin film (VTF) is a
technique in which a liquid sample is pipetted (4–6 µl) on to a transmission electron microscopy (TEM) grid and blotted to create a thin, even liquid film
across the surface of the grid. Ideally, the TEM grid should have a holey carbon film and, following blotting, the liquid sample will be held as a mono-
layer (approximately 12 nm thick) of particles supported within the holes. This film is then plunged rapidly into a liquid cryogen (ethane) to vitrify the
sample, generating a vitreous ice film that is amorphous (no long-range structure) and thus a desirable support phase for cryo-electron microscopy of
the encased particles (Cheng et al 2012). Other abbreviations: CEMOVIS, cryo-electron microscopy of vitreous sections; SEM, scanning electron
microscopy. (Adapted from Fleck RA 2015 Low-temperature electron microscopy. In: Wolkers W, Oldenhof H (eds), Methods in Cryopreservation and


Freeze-Drying Protocols. Methods in Molecular Biology. Berlin: Springer.)
nucleation of ice crystals). The nucleation of ice crystals is temperature- transferring it on to an EM grid. A double micromanipulator tool that
and pressure-dependent. Crystallization depends on the cooling rate, guides the ribbon using an electrically conductive fibre, and then posi-
itself dependent on the thermal properties of water, the sample thick- tions and attaches the ribbon to an EM grid has recently been developed
ness, and the heat extraction flow at the surface of the specimen. Freez- (Studer et al 2014) (Fig. 1.5.3).
ing is a time-dependent process. Vitrification sits at the beginning of a
workflow where the sample is either subsequently processed to a stable
Advances in instrumentation and
room-temperature state for imaging or maintained in its vitreous state
detector technology
throughout (see Fig. 1.5.2) (Fleck 2015).
There are a variety of methods for rapid freeze fixation of tissues.
However, the depth of vitrification is often limited (e.g. a few microns EM has undergone a revolution in recent years, largely coincident
for samples plunged into liquid cryogen). Of the available technolo- with the advent of digital charge-couple device (CCD) cameras and
gies, high-pressure freezing (HPF) is by far the most effective. First increasing personal computer (PC) processing power. What was princi-
introduced by Moor and Riehle in 1968, HPF exploits the physical pally a two-dimensional technique is now one that readily embraces
benefit of high pressure (210 MPa) to reduce the cooling rate required three dimensions by electron tomography (Gan and Jensen 2011).
for the vitrification of water from several 100,000 K.s−1 to a few In this technique, a specimen is tilted through the incident beam,
1,000 K.s−1, making vitrification of relatively thick samples practicable sequential images are acquired, and, after alignment, a weighted back-
(Moor and Riehle 1968, Studer et al 2008). HPF machines synchro- projection is used to generate a three-dimensional tomographic volume
nize pressurization and cooling of the sample (to below the glass (see Video 1.5.1). These techniques have stimulated instrument changes.


transition temperature (Tg)) within 20 ms, in a highly reproducible In conventional TEM, low accelerating voltages (80–120 keV) provide
manner (Müller and Moor 1984, Riehle 1968, Studer et al 2001), a strong beam-to-specimen interaction in order to increase contrast
extending the depth of vitrification to as much as 200 µm (Studer et al from electron scattering. Cryo-TEM and tomography require higher
1995, Studer et al 2008). accelerating voltages (200–300 keV), which reduce the beam-to-
Tissue samples prepared this way require further modification to specimen interaction; the contribution of chromatic aberration to the
make them compatible with the transmission electron microscope. image is also reduced, theoretically improving resolution (Williams and
They must either be converted to a room-temperature stable state (e.g. Carter 2009).
by freeze substitution into a resin), or be sectioned whilst remaining Changing the emission source has been another major technical
below the glass transition temperature (T); when this is done, images advance. A field emission gun (FEG) is becoming the preferred
g
close to the native structure of biological specimens can be achieved. source of the electron beam because it provides a beam of smaller
However, despite its benefits, cryo-electron microscopy of vitreous sec- diameter, higher coherence and a current density (brightness) of up
tions (CEMOVIS) has not been routinely adopted, principally because to three orders of magnitude greater than was achieved with conven-
of the technical difficulty of producing a ribbon of frozen sections and tional thermionic emitters (e.g. tungsten or lanthanum hexaboride


Electron microscopy in the twenty-first century
e23
5.1
YRATNEMMOC
and resolution with superior detective quantum efficiency (DQE) rela-
tive to a conventional CCD camera (Milazzo et al 2010). Moreover,
direct exposure to the incident electron beam significantly improves the
signal-to-noise ratio in comparison to an SCCD (Milazzo et al 2010).
Perhaps more importantly, DDD allow images to be recorded as movies,
which means that movement caused by the electron beam can be cor-
rected computationally (Grigorieff 2013). As an example, images of
ribosomes of sufficient quality to permit alignment at an accuracy that
approaches near-atomic resolution have been obtained using approxi-
mately 30,000 images (Bai et al 2013).
When combined, ZP, EFTEM and DDD offer a paradigm shift in the
capability of the transmission electron microscope and are likely to
drive further advances in EM-based research. Whether in isolation or
combination, these techniques will translate from cutting edge to
routine instrumentation, heralding generic benefits to the field. Phase
contrast electron microscopy utilizing ZP has already been applied
to histochemically stained strong-phase (high-contrast heavy-metal-
stained) biological specimens (Atsuzawa et al 2009). In sections of
various thicknesses specifically stained for the Golgi apparatus, a high-
contrast gain was observed even for strong-phase objects, indicating that
phase contrast electron microscopy has an application for traditional
Fig. 1.5.3 Cryo-electron microscopy of vitreous sections (CEMOVIS)
strong-phase-contrasted biological specimens (Atsuzawa et al 2009), as
performed using a cryo-ultramicrotome. The cryo-chamber (Leica EM
well as for low-phase cryo-fixed biological specimens (e.g. the visualiza-
FC6, Leica Microsystems, Austria) is mounted on a conventional
tion of cyanophage Syn5 inside its host, Synechococcus, using ZP in
room-temperature ultramicrotome (Leica EM UC6, Leica Microsystems,
combination with cryo-tomography (cryo-ET) to enhance image con-
Austria). The double manipulators (Manip, Diatome, Switzerland) are then
trast over conventional cryo-ET) and will permit direct identification of


attached to the top of the cryo-chamber, allowing precise control of
subcellular components (Dai et al 2013).
both the vitreous ribbon and the transmission electron microscopy grid.
The system is located within a glove box to maintain a low-humidity Advances in the scanning
environment around the sample that will reduce contamination during
electron microscope
sectioning.
Cryo-FEGSEM has become well established as a research tool. The sta-
bility and emitter life of a FEG source, coupled with the stability and
low keV sensitivity of a modern scanning electron microscope, is a
(LaB)-tipped filaments) (Kersker 2001). The net results for both scan-
6 powerful tool. Unlike TEM, in which high accelerating voltages are used
ning electron microscopy (SEM) and TEM have been greatly improved
to transmit the electrons through the specimen, SEM benefits from low
signal-to-noise ratio and spatial resolution, coupled with a significant
accelerating voltages that limit beam interaction with the specimen.
increase in emitter life and reliability. FEG has had a major impact on
This is key to successful cryo-SEM but creates a challenge around gen-
the practicality of cryo-SEM and resolution in cryo-TEM studies,
erating sufficient signal for high-resolution imaging. Modern FEGSEM
although these higher accelerating voltages carry a penalty of decreased
systems (with improved detector sensitivity and signal-to-noise ratios)
contrast.
are now achieving sub-nm resolution at 1 keV, making them highly
capable tools for life science research, particularly when combined with
Overcoming the challenges of contrast in
high-resolution coating and fracture techniques (Fleck 2015) (see Fig.
the transmission electron microscope
1.5.2; Fig. 1.5.4).
Zero-loss energy filtering is employed to improve image contrast in the Large three-dimensional volumes may be generated by the assembly
transmission electron microscope. The filter may be positioned either of sequential two-dimensional images acquired in the scanning elec-
in-column or post-column. Increased scattering contrast and the exclu- tron microscope. There are essentially three strategies, each with unique
sion of the blurred background (as a consequence of the interception strengths and weaknesses: focused ion beam (FIB), serial block face


of inelastically scattered electrons) allow specimens to be imaged closer SEM (SBFSEM) and array tomography (AT). FIB involves milling of
to focus, and increases high-resolution contrast and image resolution material in a dual-beam scanning electron microscope using a focused
(Grimm et al 1997, Grimm et al 1998). beam of gallium ions to cut (mill) away layers of material. After each
Energy filtered transmission electron microscopy (EFTEM) imaging cut, the surface is imaged, ultimately producing a stack of two-
to remove the contributions of inelastically scattered electrons is advan- dimensional images that can be aligned to generate a three-dimensional
tageous when imaging thick biological samples. Han et al (1995) dem- volume. Although FIB can be combined with cryo-preparation tech-
onstrated that only elastically scattered electrons contributed to the niques, elemental microanalysis and the range of imaging modes avail-
coherent image component when imaging biological specimens of able within the SEM, it can be challenging (problems include achieving
more than 0.5 µm thick at 200 keV; optimization of the zero-loss signal registry between slices, gallium contamination of the sample, and
required operation at intermediate to high primary accelerating voltages damage to structural information caused by the milling process).
(over 200 keV). The gain due to EFTEM increases with specimen thick- SBFSEM takes a different approach from FIB and incorporates a
ness, with a linear dependence for light-scattering elements (Grimm microtome within the scanning electron microscope chamber (Denk
et al 1998). These results are important for the accurate recording of and Horstmann 2004). The microtome cuts a thin section from the
images of thick biological specimens by tomography and to demon- block, an image is taken, and the procedure is repeated until a three-
strate the increasing importance of high accelerating voltages in biologi- dimensional volume is generated (Video 1.5.2). SBFSEM is less prone
cal TEM (Grimm et al 1998). to registration issues because the sample essentially remains locked in
New technologies are overcoming previous limitations of low con- position below the detector. However, like FIB, this technique is destruc-
trast in the transmission electron microscope. Zernike phase plates tive since, once sectioned, the sample is lost. SBFSEM uses room tem-


(ZP), which are well accepted for imaging with the light microscope, perature processing techniques with high levels of en bloc heavy metal
represent a novel approach to imaging in the transmission electron staining that are required to generate a sufficiently robust back-scattered
microscope. Despite technical hurdles to their use, the potential reward electron signal to form an image. The processing steps, and the limita-
in greater contrast has driven research into this technology (Fukuda tions of the back-scattered electron detector, mean that SBFSEM is cur-
et al 2009, Glaeser 2013). An approximate five-fold increase in contrast rently incompatible with elemental microanalysis or cryo-techniques.
is possible with a π ZP that shifts the phase of only the scattered elec- However, this is a nascent technology that is advancing rapidly. Novel
2
trons (these phase-shifted electrons interfere with the unscattered elec- applications will emerge with advances in detectors, improved resins
trons in the image plane), resulting in amplitude contrast rather than (e.g. conductive resins) and the exploitation of charge suppression
phase contrast. Indirect scintillator-coupled charge-coupled-device approaches in the SEM (e.g. beam deceleration).
(SCCD) cameras replaced film cameras and revolutionized EM because Unlike FIB and SBFSEM, AT is not destructive and material may be
they provided a near-instant digital readout and obviated the physical recovered and re-imaged. The technique allows samples to be prepared
limitation of a fixed number of image plates. Direct detection devices by any TEM preparation strategy. Material is sectioned at room tempera-
(DDD) avoid the fundamental inefficiencies of the electron-to-light ture by ultramicrotomy; the sections are collected in order as a ribbon
conversion process of a scintillator and achieve unmatched sensitivity and may then be stained to enhance contrast, or are immunolabelled.


ElECTRON MiCROsCOpY iN ThE TwENTY-fiRsT CENTuRY
e24
1
NOiTCEs
A B
Fig. 1.5.4 Cryo-scanning electron microscopy (SEM) images. A, Fractured Plasmodium falciparum-infected erythrocytes. A cross-fractured infected
erythrocyte is shown; the insert depicts a high-magnification image of the p-face of the infected erythrocyte with membrane proteins visible on the
fracture plane. The scale bar represents 1 µm (main image) and 100 nm (inset). B, Cross-fractured Neisseria meningitidis showing surface membrane
protein organization and polysaccharide coat. The scale bar represents 100 nm. Images were taken with a cold-field emission gun cryo-SEM (JSM-
7401F, JEOL, Japan), equipped with a Baltec/Leica VCT100 vacuum transfer system and cryo-stage. Coating was applied after fracturing (3 nm Pt/C)
using multi-angle rotational coating (BAF060, Baltec/Leica Microsystems, Austria).
Ribbons may be imaged by LM or SEM; individual sections can be
identified, recovered and imaged at the higher spatial resolution of the
transmission electron microscope. Successful AT requires high-precision
x-y-z sample stages that can perform programmed, sequential (in focus)
imaging of serial sections. Arguably the most challenging aspect of the
workflow is the serial sectioning of the sample; to date, this is under-
taken almost exclusively by highly skilled operators. However, the
ATUMtome (RMC, USA) employs a tape system to automate the serial
sectioning process and has recently been commercialized (Fig. 1.5.5).
New multibeam-multidetector SEMs (e.g. 61 concurrent beams) are
being developed that will allow images of up to 1.2 billion pixels to be
acquired in 1 s (Marx 2013) and will further speed data acquisition.
Developments in thin-film technology and microfabrication are
facilitating the design of liquid cells that will permit improved imaging
of samples in the liquid phase (de Jonge and Ross 2011). In combina-
tion with recent advances in ZP, EFTEM and DDD, the new generation


of liquid cells offers considerable potential for the study of complex
dynamic cellular processes.
Fig. 1.5.5 ATUMtome and tape. The ATUMtome is mounted on an PT-PC
PowerTome (RMC, USA), showing the tape being transferred from a
delivery spool (top) to receiving spool (bottom) through the boat, which
maintains a water reservoir behind the edge of the diamond knife (blue) to
float the sections and allow their capture by tape. Tape tension is
maintained throughout by a series of tensioning springs and guides. The
insert shows individual sections on tape mounted on carbon tape and
silicon wafer (to help dissipate specimen charging during imaging) ready
for viewing in the scanning electron microscope.
REFERENCES
Atsuzawa K, Usuda N, Nakazawa A et al 2009 High-contrast imaging of Dai W, Fu C, Raytcheva D et al 2013 Visualizing virus assembly intermediates
plastic-embedded tissues by phase contrast electron microscopy. J Elec- inside marine cyanobacteria. Nature 502:707–10.
tron Microscopy 58:35–45. de Haro T, Furness P 2012 Current and future delivery of diagnostic electron
Bai XC, Fernandez IS, McMullan G et al 2013 Ribosome structures to near- microscopy in the UK: results of a national survey. J Clin Pathol 65:
atomic resolution from thirty thousand cryo-EM particles. Elife 2:e00461. 357–61.
Boyde A, Maconnachie E 1979 Volume changes during preparation of de Jonge N, Ross F 2011 Electron microscopy of specimens in liquid. Nat
mouse embryonic tissue for scanning electron microscopy. Scanning Nanotechnol 6:695–704.
2:149–63. Denk W, Horstmann H 2004 Serial block-face scanning electron microscopy
Cheng D, Mitchell DRG, Shieh D-B et al 2012 Practical considerations in to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:
the successful preparation of specimens for thin-film cryo-transmission e329.
electron microscopy. In: Méndez-Vilas A (ed) Current Microscopy Con- Fleck RA 2015 Low-temperature electron microscopy. In: Wolkers W,


tributions to Advances in Science and Technology, Microscopy Series no. Oldenhof H (eds), Methods in Cryopreservation and Freeze-Drying,
5 (vol. 2). Badajoz: Formatex; pp. 880–90. Lab Protocol Series, Methods in Molecular Biology. Berlin: Springer;
Claeys M, Vanhecke D, Couvreur M et al 2004 High-pressure freezing and pp. 243–74.
freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhab-
ditida) for transmission electron microscopy. Nematology 6:319–27.


Electron microscopy in the twenty-first century
e25
5.1
YRATNEMMOC
Fukuda Y, Fukazawa Y, Danev R et al 2009 Tuning of the Zernike phase-plate Müller M, Moor H 1984 Cryofixation of thick specimens by high pressure
for visualization of detailed ultrastructure in complex biological speci- freezing. In: Revel JP, Barnard T, Haggis GH (eds) Science of Biological
mens. J Struct Biol 168:476–84. Specimen Preparation. AMF O’Hare, Chicago: SEM; pp. 131–8.
Gan L, Jensen GJ 2011 Electron tomography of cells. Q Rev Biophys Nermut MV 1991 Unorthodox methods of negative staining. Micron Microsc
45:27–56. Acta 22:327–39.
Glaeser RM 2013 Methods for imaging weak-phase objects in electron Nermut MV, Frank H 1971 Fine structure of Influenza A2 (SINGAPORE) as
microscopy. Rev Sci Instrum 84:111101. revealed by negative staining, freeze-drying and freeze-etching. J Gen
Glauert AM, Lewis PR 1998 Biological specimen preparation for transmis- Virol 10:37–51.
sion electron microscopy. London: Portland Press; p. 326. Plitzko JM, Rigort A, Leis A 2009 Correlative cryo-light microscopy and
Grigorieff N 2013 Structural biology: direct detection pays off for electron cryo-electron tomography: from cellular territories to molecular land-
cryo-microscopy. Elife 2:e00573. scapes. Curr Opin Biotechnol 20:83–9.
Grimm R, Bärmann M, Häckl D et al 1997 Energy-filtered electron tomog- Riehle U 1968 Über die Vitrifizierung verdünnter wässriger Lösungen. Dis-
raphy of ice-embedded actin and vesicles. Biophys J 72:482–89. sertation No. 4271: Eidgenössiche Technische Hochschule. Zurich:
Federal Institute of Technology (ETH).
Grimm R, Typke D, Baumeister W 1998 Improving image quality by zero-


loss energy filtering – quantitative assessment by means of image cross- Robards AW, Sleytr UB 1995 Low temperature methods in biological elec-
correlation. J Microsc-Oxford 190:339–49. tron microscopy. In: Glauert AM (ed.), Practical Methods in Electron
Microscopy, vol. 10. Amsterdam: Elsevier; pp. 5–146.
Han KF, Sedat JW, Agard DA 1995 Mechanism of image formation for thick
biological specimens: exit wavefront reconstruction and electron energy- Sartori A, Gatz R, Beck F et al 2007 Correlative microscopy: bridging the gap
loss spectroscopic imaging. J Microsc-Oxford 178:107–19. between fluorescence light microscopy and cryo-electron tomography.
J Struct Biol 160:135–45.
Hayat MA 2000 Chemical fixation. In: Principles and Techniques of Electron
Microscopy: Biological Applications, 4th ed. Cambridge: Cambridge Steinbrecht RA, Zierold K 1987 Cryotechniques in biological electron micro-
University Press, 4–80. scopy. Berlin: Springer; p. 293.
Kellenberger E 1991 The potential of cryofixation and freeze substitution: Studer D, Graber W, Al-Amoudi A et al 2001 A new approach for cryofixation
observations and theoretical considerations. J Microsc-Oxford 161: by high-pressure freezing. J Microsc-Oxford 203:285.
183–203. Studer D, Humbel BM, Chiquet M 2008 Electron microscopy of high pres-
Kersker MM 2001 The modern microscope today. In: Zhang X-F, Zhang Z sure frozen samples: bridging the gap between cellular ultrastructure
(eds) Progress in Transmission Electron Microscopy 1: Concepts and and atomic resolution. Histochem Cell Biol 130:877–89.
Techniques. Berlin: Springer; pp. 1–34. Studer D, Klein A, Iacovache I et al 2014 A new tool based on two micro-
McDonnald KL 2009 A review of high-pressure freezing preparation tech- manipulators facilitates the handling of ultrathin cryosection ribbons.
niques for correlative light and electron microscopy of the same cells J Struct Biol 185:125–8.


and tissues. J Microsc-Oxford 235:273–81. Studer D, Michel M, Wohlwend M et al 1995 Vitrification of articular carti-
Marx V 2013 Brain mapping in high resolution. Nature 503:147–52. lage by high-pressure freezing. J Microsc-Oxford 179:321–32.
Milazzo AC, Moldovan G, Lanman J et al 2010 Characterization of a direct Williams DB, Carter CB 2009 Transmission Electron Microscopy: A Text-
detection device imaging camera for transmission electron microscopy. book for Materials Science. Berlin: Springer; p. 760.
Ultramicroscopy 110:744–7.
Moor H, Riehle U 1968 Snap-freezing under high pressure: A new fixation
technique for freeze-etching. Proc Fourth Europ Reg Conf Elect Microsc
2:33–4.


e26
1
NOITCES
COMMENTARY
The reaction of peripheral nerves
1.6
to injury
Rolfe Birch
Main nerve trunks contain many thousands of axons. When a trunk is Transient ischaemia Lying in one position without moving for a
stretched, but not ruptured, by dislocation of, say, the femoral or long time (e.g. during coma, inebriation or anaesthesia), prolonged
humeral heads, or by the nearby passage of a bullet, the axons within sitting with the legs crossed or prolonged leaning on the elbows are all
that nerve may respond in different ways. The degree of injury may vary situations in which a limb nerve may be transiently compressed against
along the length of a particular nerve or transversely across its cross- a bone or a hard surface. The ischaemia thus induced in the compressed
section: some axons will remain intact, some will sustain conduction nerves causes an anoxic, physiological, block of both axoplasmic trans-
block but recover rapidly, and yet others will degenerate throughout port and ion channel functions along affected axons. This is seen during
their entire extent distal to the injury because they have been ruptured. operation for the exposure of limb nerves with an inflated cuff in posi-
A proportion recover spontaneously whilst the remainder never recover. tion: stimulation of a nerve evokes a brisk muscular response by trans-
Seddon (1943) introduced the terms neurapraxia for conduction mission through the neuromuscular junction, which then diminishes
block, axonotmesis for a degenerative lesion of favourable prognosis, before disappearing after about 30 minutes, whereas conduction within
and neurotmesis for a degenerative lesion of unfavourable prognosis. the nerve itself can be detected for about another 30 minutes. On the
Sunderland (1951) expanded this classification to five degrees of other hand, direct stimulation of the muscle provokes a twitch that can
peripheral nerve injury by subdividing neurapraxia according to the be elicited for about another 2 hours; loss of this direct response signals
degree of connective tissue disruption, and MacKinnon and Dellon impending muscle death. The selective vulnerability of different popu-
(1988) subsequently added a sixth category. However, the fundamental lations of nerve fibres is demonstrated by the classical experiment of


distinction between neurapraxia and either axonotmesis or neurotmesis applying an inflated cuff about the arm (Lewis et al 1931). The observer
remains the persistence of conduction in the distal segment of the nerve experiences first a loss of superficial sensibility, then a graduated loss
in neurapraxia, and the absence of conduction in axonotmesis or neu- of muscle power. The first pain response is lost soon after superficial
rotmesis. In clinical practice, neurotmesis usually signifies transection sensibility fails; the delayed pain response is still detectable after
of an entire nerve trunk, its constituent fascicles and their surrounding 40 minutes of ischaemia. Large myelinated axons are first affected;
investments of perineurium and epineurium; the proximal and distal unmyelinated axons and autonomic fibres escape; and pilo- and vaso-
nerve stumps are frequently separated by an inter-stump gap of varying motor functions are scarcely affected. All modalities recover within a
length (Fig. 1.6.1). The early disappearance of conduction indicates few minutes of release of the cuff; the unpleasant quality of the residual
impending or actual critical ischaemia and is probably the most impor- delayed pain sensation, and the burst of painful ‘pins and needles’ after
tant indicator that a lesion is deepening (see below). release of the cuff, give an insight into the feelings of patients affected
by dysaesthesiae.
Neurapraxia/conduction block
Slowly progressing ischaemia Conduction block caused either
Conduction block exhibits certain characteristic features: paralysis by a haematoma or aneurysm, or by bleeding into compartments pro-
exceeds loss of sensation; proprioceptive nerves are more deeply affected duces early and deep autonomic paralysis and loss of power that
than those responsible for light touch; and sympathetic nerves to the extends over hours or days, whilst deep pressure sense and some joint
smooth muscles of the blood vessels and sweat glands are least affected. position sense persist. These symptoms are seen in war wounds, when
There are distinct patterns of conduction block. Anoxia is important in nerves become compressed and strangled by scar tissue or by the cicatrix
all of them and there are often elements of mechanical deformation, deep to split skin grafts, and may progress over a period of weeks or


inducing alterations of the nodal and paranodal apparatus in myeli- months. The recovery of function and relief of pain are rapid – at times,
nated axons, and subsequent demyelination either of whole internodes dramatic – after removal of the cause in most of these cases.
or of paranodes.
Prolonged conduction block associated with focal demyel
ination Severe prolonged pressure causes local demyelination and a
conduction block that is not directly attributable to anoxia and that
Basal lamina Schwann cell nucleus
may last for weeks or months (Fig. 1.6.2). Compression-induced focal
paranodal myelin deformation may be involved; experimental studies
using a cuff inflated to high pressure around a limb produced slippage
of paranodal myelin at nodes of Ranvier lying under the edges of the
cuff, i.e. where the pressure gradient between compressed and non-
compressed portions of the nerve was likely to be greatest (Ochoa et al
1972, Dyck et al 2005). Clinically, local demyelination and conduction
Node of Ranvier Myelin sheath Axon
Proximal Cuff Distal
Axon
Fig. 1.6.1 Axonotmesis (centre) and neurotmesis (bottom) at the moment Fig. 1.6.2 The effect of mechanical compression: paranodal and
of injury. Note the preservation of the Schwann cell basal lamina in juxtaparanodal myelin slippage under the edges of an inflated pressure
axonotmesis. (Courtesy of Rolfe Birch, all rights reserved, published in cuff (after Ochoa J, Fowler TJ, Gilliatt RW 1972). (Courtesy of Rolfe Birch,
Birch R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011. all rights reserved, published in Birch R, Surgical Disorders of the
Springer-Verlag, London.) Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, London.)


The reaction of peripheral nerves to injury
e27
6.1
YRATNEMMOC
Fig. 1.6.4 The appearance of a radial nerve after extrication from beneath
Fig. 1.6.3 Hourglass ‘constriction’ of the lateral root of the median nerve: a compression plate 48 hours after the first operation. There was relief of
the result of traction injury. There was full spontaneous recovery. pain but only incomplete recovery so that subsequent flexor to extensor
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical transfer was necessary. (Courtesy of Rolfe Birch, all rights reserved,
Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd
London.) edition, 2011. Springer-Verlag, London.)
block will persist until the source of the external irritation, e.g. a bony
projection, is removed (Birch and St Clair Strange 1990). Relief of pain
and recovery follow rapidly in lesions that have persisted for many
months, or even years. Mechanical deformation is the likely explanation
for the conduction block seen in cases of ‘hourglass’ constriction of
fascicles within a nerve trunk, where axons may be focally constricted
within affected fascicles (Fig. 1.6.3).
Prolonged conduction block of war wounds The characteristic
features of classical neurapraxia (see above), caused by the nearby
passage of a missile, are likely to be provoked by a momentary displace-
ment or stretching of the nerve trunks. However, a different pattern of
conduction block has been recognized in recent conflicts, where the
patient is exposed, at close range, to the shock wave of an explosion
without any wound or fracture, or signs of significant injury to the soft
tissues at the level of the nerve lesion. In these cases, the smallest fibres
are most deeply affected and they may not recover. Pain is rare in both A
of these variants of conduction block (Birch et al 2012).
Degenerative lesions
There are essentially two types of degenerative lesion, with very different
potentials for recovery. In the first, which approximates to axonotmesis,
the tubes of Schwann cell basal lamina that surround each axon–


Schwann cell unit remain intact, which means that axons may regener-
ate in an orderly fashion across the lesion site and into the distal stump,
providing that the causative agent is removed and that the length of the
distal stump (i.e. the distance from the site of injury to the end organ)
is not so long that it compromises axonal regrowth. In the second,
which approximates to neurotmesis, not only the Schwann cell basal
lamina tubes, but also the connective tissue sheaths around the fascicle B
are interrupted; spontaneous axonal regeneration following this type of
injury will be imperfect and disorderly, or may not occur at all.
Fig. 1.6.5 Volkmann’s ischaemic contracture. A, The ulnar nerve exposed
Deepening of a lesion in a nerve that has during flexor muscle slide 8 weeks after supracondylar fracture. The
epineurial vessels and also the ulnar recurrent collateral vessels are
not been divided
occluded, and the nerve is compressed by the swollen infarcted muscle.
Unrelieved activity of a noxious agent on an intact nerve is signified by B, The appearance of the hand 14 years later. (Courtesy of Rolfe Birch, all
pain and increasing neurological deficit, indicating that the lesion is rights reserved, published in Birch R, Surgical Disorders of the Peripheral
deepening. Nerves within a swollen ischaemic limb or tense compart- Nerve. 2nd edition, 2011. Springer-Verlag, London.)
ment become compressed and anoxic. Nerves displaced by an expand-
ing haematoma become stretched and anoxic. Nerves that have become 1.6.5). (iii) A nerve displaced into a fracture or joint will usually recover
displaced into a fracture or joint are tethered and become subjected to if it is extricated within a few days, but will be destroyed if a compres-
compression, stretching and anoxia. These nerves are not severed by the sion device has been used to stabilize the skeleton without retrieving
initial injury; the first lesion is axonotmesis and spontaneous recovery the nerve (Fig. 1.6.6). (iv) Function may be retained for a long time in


is probable if the cause is removed. However, if this does not happen, a nerve exposed to continuing traction from a malunited fracture. (v)
the lesion continues to deepen into that of neurotmesis and spontan- Nerves may continue to work for months or years before failing when
eous recovery will not occur. The speed of deepening of a lesion is they are exposed to the progressing fibrosis caused by radiotherapy, by
related to the cause: (i) A nerve crushed by a plate or strangled by an overlying split skin graft or by other examples of deep scar. (vi) Pain is
encircling suture may recover if the cause is removed within a few the cardinal symptom of the unrelieved action of a noxious agent.
minutes (Fig. 1.6.4). (ii) A nerve that has become compressed and
anoxic within a swollen ischaemic limb will recover if perfusion of the Wallerian degeneration
tissues is restored within 3 hours; the likelihood of full spontaneous Trauma that physically separates axons from their cell bodies produces
recovery diminishes with the passage of every hour after that time (Fig. Wallerian degeneration (WD). All affected axons in the extreme tip of


ThE REACTION Of pERIphERAl NERvES TO INjuRY
e28
1
NOITCES
involved in retrograde signalling to initiate expression of RAGs, particu-
larly the family of glycoprotein 130 (gp130) cytokines, see Zigmond
(2011).) Changes in the expression of ion channels and receptors in the
neuronal cell bodies in human DRGs can be detected within a few
minutes of an injury to the nerve (Lawson 2005).
Tip of proximal stump Within a few hours of injury, the cut ends
of a transected axon seal off, forming end bulbs. The end bulb that
forms at the proximal tip of axons in the proximal stump is transformed
into a growth cone from which multiple needle-like filopodia and
broader sheet-like lamellipodia grow out. The filopodia are rich in
actin, and may extend or retract within a matter of minutes. Each axon
forms new branches or sprouts: collateral sprouts arise from nodes of
Ranvier at levels where the axons are still intact; terminal sprouts arise
from the tips of the surviving axons. The axon sprouts and fine cyto-
A plasmic processes derived from their associated Schwann cells form
clusters, regenerating units, surrounded by Schwann cell basal laminae.
Sprouts within one regenerating unit represent the regenerative effort
of one neurone and its axon. Within a few days of injury, the calibre of
axons in the proximal stumps is reduced and nerve conduction velocity
in the proximal segment falls.
Distal stump During the first 2 or 3 weeks after injury, the constitutive
tissue response within a denervated distal stump involves numerous
events: some sequential, others consecutive. These include the produc-
tion of debris, as axons and their myelin sheaths are degraded; an
increase in local blood flow; activation of resident macrophages, and
recruitment and influx of exogenous macrophages; and proliferation of
fibroblasts and Schwann cells. The gliotic response produces a popula-
tion of dedifferentiated daughter Schwann cells that remain within the
basal lamina tubes secreted by their parent cells, forming what are now
called Schwann tubes but which were previously known as bands of


Büngner. They behave acutely as the ‘presumed targets’ of the regrowing
B
axons until the actual targets (muscle or sensory end organ) have been
reinnervated: they secrete proteins that collectively facilitate axonal
Fig. 1.6.6 Deepening of a lesion. A, A median nerve extricated from a regrowth, whether by providing trophic support or by establishing a
supracondylar fracture in a 9-year-old girl 3 days after injury; there was supportive growth matrix (for further reading on these interactions, see
complete recovery. B, A median nerve extricated from a supracondylar Hall (2005), Webber et al (2011), Arthur-Farraj et al (2012), Gordon
fracture in a 13-year-old girl 8 weeks after injury; there was no recovery. (2015)). There is a growing consensus that Schwann cells express dis-
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical
tinct motor and sensory phenotypes, and that this fundamental differ-
Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag,
ence affects the ability of Schwann cell tubes to selectively support
London.)
regenerating neurones. For example, expression of osteopontin and
clusterin is upregulated in Schwann cells in transected peripheral
nerves: the two secreted factors appear to facilitate regeneration of
the proximal stump and throughout the distal stump degenerate, irre- motor or sensory neurones, respectively (Wright et al 2014).
spective of their calibre or functional modality; recovery is often slow While the microenvironment of an acutely denervated distal stump
and incomplete, and there may be considerable residual loss of func- facilitates axonal regrowth because it provides a vascularized segment
tion. Moreover, although research has tended to address issues associ- of longitudinally orientated, laminin-rich basal lamina tubes filled with
ated with the molecular and cellular events that occur at the lesion site axon-responsive Schwann cells, a chronically denervated distal stump
and target tissues, it is important to remember that injury to a periph- is associated with poor axonal regeneration and poor functional recov-


eral nerve that induces WD also initiates significant and often long- ery (Sulaiman and Gordon 2009). Biopsies taken during late repairs,
lasting central changes in sensory ganglia and ascending and descending especially when the injury has been complicated by arterial injury or
pathways within the spinal cord and brain; these changes also have a by sepsis, often show remarkably few Schwann cells lying within a
significant impact on the likelihood of recovery. Studies of cell bodies densely collagenous matrix. Myelin fragments are detectable in such
of human dorsal root ganglia (DRG) have revealed changes in the cases many months after the injury. The residual Schwann cells become
expression of a wide range of receptors and a remarkable alteration in progressively less receptive to ingrowing regenerating axons with time;
the expression of genes regulating neuronal activity (Lawson 2005, experimental studies have shown that they downregulate expression of
Rabert et al 2004, Anand et al 2008). There is an extensive literature on axon-responsive receptors that are normally important in Schwann
WD in both central and peripheral nervous systems, and only a brief cell–axon signalling (Li et al 1997, Li et al 1998). These findings under-
summary of the response in the peripheral nervous system is presented score the clinical observation that there is a relatively narrow window
here (see Gordon (2015)). of opportunity when surgical intervention is most likely to produce
positive results (Lundborg 2000).
Neuronal cell body and proximal stump The central and the
The special case of preganglionic injury
peripheral effects of WD are profound and, in neurotmesis, ultimately
irreversible. Not only is the neuronal cell body separated from its con- The preganglionic lesion is all too common in severe traction injuries
tinual supply of retrogradely transported neurotrophins, but also its to the brachial and lumbosacral plexuses. The roots of the spinal nerves
central connections rapidly alter; many neurones thus isolated will die. are torn from the spinal cord, which means that the somatic afferent
Amputation provides a model of the effect of permanent axonotomy pathway is interrupted between the dorsal root ganglion and the spinal
on the spinal cord: there is extensive loss of neurones in the dorsal root cord (see Fig. 1.6.6). The neuronal cell bodies and their axons, investing


ganglia and in the anterior horn, and a diminution of the large myeli- Schwann cells and associated basal laminae remain intact, healthy and
nated axons in the ventral and dorsal roots (Dyck et al 1984, Suzuki conducting for a long time after the injury. The surviving axons include
et al 1993). Neuronal death is more severe in more proximal neurot- all those with cell bodies in the dorsal root ganglion, including many
mesis, and less marked after axonotmesis than after neurotmesis. Neu- ‘recurrent’ fibres in the ventral root that derived from cells in the dorsal
rotmesis in the neonate produces a more rapid and much greater root ganglion (Figs 1.6.7–1.6.9). Somatic efferent fibres, being sepa-
incidence of sensory and motor neurone death than in the adult. Surviv- rated from their cell bodies, degenerate; postganglionic autonomic
ing neurones rapidly upregulate a series of genes involved in repair and efferent axons also degenerate, as a result of damage to their grey rami
neuroprotection mechanisms following injury (repair-associated genes, communicantes. Carlstedt (2007) reckons that about one-half of all
RAGs), which direct many of the changes seen during WD and subse- motor neurones in the affected spinal cord segment have disappeared
quent recovery (e.g. Calenda et al 2012). (For a review of the molecules by 2 weeks after avulsion of the ventral root and he urges ‘a swift


The reaction of peripheral nerves to injury
e29
6.1
YRATNEMMOC
Fig. 1.6.7 Intact
(afferent) myelinated
and unmyelinated fibres
in a suprascapular
nerve, 6 weeks after an
avulsion lesion of the
brachial plexus;
efferent fibres have
degenerated.
Magnification ×6600.
(Courtesy of Rolfe
Birch, all rights
reserved, published in
Birch R, Surgical
Disorders of the
Peripheral Nerve. 2nd
edition, 2011. Springer-
Verlag, London.)
Fig. 1.6.9 Afferent and efferent fibres in a ventral root. Healthy myelinated
and unmyelinated fibres in the ventral root of the eighth cervical nerve
avulsed from the spinal cord 6 weeks previously. The myelinated efferent
fibres have undergone Wallerian degeneration and there is a notable
increase in the amount of endoneurial collagen. (Courtesy of Rolfe Birch,
all rights reserved, published in Birch R, Surgical Disorders of the
Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, London.)
Regeneration of end organs
Reinnervation of muscle spindles and Golgi tendon receptors is gener-
ally good after crush lesions (axonotmesis). Regeneration after repair
of a divided nerve is much less orderly. A muscle spindle may become
reinnervated by afferents normally destined for the Golgi tendon organ;
many tendon organs remain denervated and the regenerated endings
are frequently abnormal in appearance. The reorganization of motor
units after repair causes significant alterations in the mechanical input
to an individual tendon organ.
Muscles usually exhibit weakness, impaired coordination and
reduced stamina after nerve repair. There may be selective failure of
regeneration of the largest-diameter fibres and of coactivation of the α
and γ efferents. The normal movement of joints is brought about by
smoothly coordinated and controlled activity in muscles that is pre-
cisely and delicately regulated by inhibition and facilitation of the
motor neurones. The conversion of an antagonist to an agonist is the
basis of musculotendinous transfer; it is common to see patients


Fig. 1.6.8 Wallerian degeneration in the ventral root of the eighth cervical
nerve, 6 weeks after avulsion from the spinal cord. A degenerate efferent actively extending the knee or the ankle and toes as soon as the post-
myelinated fibre (right) compared with a healthy myelinated afferent fibre operative splint is removed after hamstring to quadriceps transfer or
(left). Magnification ×11115. (Courtesy of Rolfe Birch, all rights reserved, anterior transfer of tibialis posterior. Reinnervated muscles usually fail
published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd to convert after muscle transfer, irrespective of their power. Perhaps the
edition, 2011. Springer-Verlag, London.) defective reinnervation of the deep afferent pathways from the muscle
spindles and the tendon receptors blinds muscles, which are, after all,
sensory as well as effector organs.
intervention to re-establish contact between the injured nerve cells and Cutaneous sensory receptors similarly undergo a slow degenerative
the periphery with its supply of neurotrophic substances to counteract change after denervation and after 3 years they may disappear. Re-
nerve cell loss’. After avulsion injury, human dorsal root ganglion neur- innervation tends to reverse these changes, although the longer the
ones show dramatic changes in the expression of genes involved in period of denervation, the less complete will be the regeneration. It is
neurotransmission, trophism, cytokine function, signal transduction, usual to find numerous myelinated axons in the tissues bridging nerve
myelination, transcription regulation and apoptosis (Rabert et al 2004). stumps in the human, even in failed sutures or in grafts coming to revi-
sion (Fig. 1.6.10) (Terenghi et al 1998); these findings encapsulate the
Regeneration and recovery of function difference between regeneration and recovery of function.
Plasticity in the central nervous system
‘But the journey of the axon tip to an end organ is only the most
dramatic of the phases in the process of regeneration, and its arrival is
alone no guarantee of the return of useful function.’ Nerve transfer involves connecting the proximal stump of a healthy
nerve to the distal stump of one that has been injured in such a way


Young (1942) that direct repair is not possible.
The cellular microenvironment of a normal peripheral nerve does not There is considerable adaptation of the central receptor and effector
ordinarily support axonal regrowth; indeed, there appear to be ‘molecu- mechanisms after nerve transfer (Fig. 1.6.11). Independent flexion of
lar brakes’ that prevent axonal sprouting. Somewhat counterintuitively, the elbow without associated activity in the flexor muscles of the
it appears that their expression may be upregulated after injury. WD forearm is usual by 24 months after transfer from the ulnar nerve to the
transforms the environment throughout the distal stump of a transected nerve to biceps brachii. Reinnervation of the muscle spindles and cor-
nerve to one that facilitates axonal regrowth, albeit for a relatively short responding reorganization and remapping of the somatosensory cortex
period (Christie and Zochodne 2013). Manipulating the many cellular have been confirmed after transfer of intercostal nerves to the musculo-
and molecular responses that occur during the injury response in the cutaneous nerve in adult patients with severe lesions of the brachial
peripheral nervous system remains an as yet unachieved goal of recon- plexus (Malessy et al 2003, Sai et al 1996). These central phenomena
structive surgery. may explain the remarkably good recovery of sensation within the hand


ThE REACTION Of pERIphERAl NERvES TO INjuRY
e30
1
NOITCES
C8
via
XI
SS C5
C5
c5 RH Age 6y
c6
c7 C6 Normal
Lat. CW, PP, VIB
JPS, CS, WS
C7 localization
C8
T1 C6, C7
C8 via
T3–T5
T3
T1
T4
T5
Fig. 1.6.12 The method of repair and the likely pathways for afferent
Fig. 1.6.10 Useless regeneration in a median nerve sutured 3 years function. Abbreviations: CS, cool sensation; CW, cotton wool; JPS,
previously. Proximal to the suture line, the endoneurium contains thinly joint position sense; PP, pinprick; Vib, vibration threshold; WS, warm
remyelinated axons, some in regenerating clusters, and extensive sensation. (Courtesy of Rolfe Birch, all rights reserved, published in Birch
collagenization; there was no recovery of function. Magnification ×4300. R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011.
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical Springer-Verlag, London.)
Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag,
London.)
Fig. 1.6.11 A 13-year-old boy with a right-sided lesion: avulsion of C5, Fig. 1.6.13 Birth lesions of the brachial plexus. This boy had a group 4
6 and 7. Repair was undertaken at 2 months as follows: accessory to lesion with rupture of C5 and avulsion of C6, 7, 8 and T1 at birth. At
suprascapular transfer; one bundle of the ulnar nerve to the nerve to 10 weeks of age, C5 was grafted to the upper trunk and to the ventral
biceps and the nerve to the medial head of triceps; medial cutaneous root of C8 and T1; accessory nerve was transferred to the ventral root


nerve of the forearm to the lateral root of the median nerve. Results at of C7; the sensory divisions of intercostal nerves T3 and T4 were
18 months show a full range of lateral rotation; abduction to 60°; power of transferred to the lateral root of the median nerve. He regained a useful
elbow flexion, Medical Research Council (MRC) grade 4; power of elbow grasp between thumb and index finger, and is an extremely well-adjusted
extension, MRC grade 3+. There is a full range of active flexion and sportsman and musician. The photograph was taken at 10 years of age.
extension without obvious co-contraction. (Courtesy of Rolfe Birch, all (Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical
rights reserved, published in Birch R, Surgical Disorders of the Peripheral Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag,
Nerve. 2nd edition, 2011. Springer-Verlag, London.) London.)
after repair of severe birth lesions of the brachial plexus. Indeed, the whilst afferent impulses from the little and, probably, the ring fingers
sensory recovery is far better than that of skeletal muscle and sympa- entered the spinal cord through the fifth cervical nerve as shown in
thetic function. Thus, there is accurate localization to the dermatomes Figure 1.6.12. Similar findings have been observed in four other cases
of avulsed spinal nerves that had been reinnervated by intercostal nerves after transfer of intercostal nerves to the lateral cord (Fig. 1.6.13). The
transferred from remote spinal segments (Anand and Birch 2002). situation in the adult is very different. After transfer of the intercostal
nerves to the lateral or medial cords, it is usual to find that stimulation
Case report
of the reinnervated skin is referred to the chest wall and only rarely to
Operation in a 6-month-old boy confirmed rupture of C5 with avulsion the skin of the hand (Htut et al 2006). Concepts of the mechanisms
of C6, 7, 8 and T1. The lesion was repaired by transfers of the accessory underlying cortical plasticity following nerve injury and the importance


nerve to the suprascapular nerve, of C5 to C8 and T1, and of intercostal of maintaining an active sensory map of the affected part in the somato-
nerves T3, 4 and 5 to the lateral cord of the brachial plexus. At the age sensory cortex during the deafferentation period are considered further
of 5 years, he was able to localize cotton wool and pinprick sensation by Lundborg (2003), Rosén and Lundborg (2007), Björkman et al
accurately in both hands and was able to feel a pin in the affected side. (2009), Rosén et al (2011) and Knox et al (2015).
Monofilament, vibration thresholds and joint position sense were iden-
tical in both hands. The thresholds for warming were 3.1°C (C5), 0.8°C
Laboratory and clinical studies
(C6), 4.1°C (C7), 3.6°C (C8) and 3.3°C (T1) on the affected side. The
thresholds for cooling were 2°C (C5), 3.9°C (C6), 3.8°C (C7), 2.3°C
(C8) and normal in T1. In the unaffected hand, the thresholds were There are major differences between a laboratory investigation, in
less than 3.4°C for warm sensation and less than 2.2°C for cool sensa- which a controlled, precise and limited lesion is inflicted on a periph-
tion. Sweating was 70% of that of the intact palm. It seems likely that eral nerve in an anaesthetized experimental animal, and the situation
afferent impulses from the thumb, index and, probably, the middle faced by the clinician presented with a patient with a massive wound
fingers entered the spinal cord through the three intercostal nerves, involving soft tissues (including blood vessels and muscles) and skeletal


The reaction of peripheral nerves to injury
e31
6.1
YRATNEMMOC
elements, and sometimes with other injuries that threaten life and limb. As time goes by, the cellular response in both stumps changes from
That said, the fundamental cellular processes that underlie subsequent one that facilitates regeneration to one that is less favourable. Dense
regeneration are similar in the laboratory, in the injuries of civilian collagenization, a profusion of fibroblasts and a loss of Schwann cells
practice and in the wounds of war, although in clinical practice they are are characteristics of the chronically denervated distal stump and are
modified by the violence of the injury, by the effects of injury on associ- typical of late cases.
ated tissues and, in particular, by ischaemia and by delay before repair. The normal architecture of a nerve is most closely restored to normal
Some general conclusions may be drawn from extensive clinical and in a well-executed, tension-free primary suture.
laboratory studies. Regeneration through a graft falls away along its length and not
The concept of retrograde degeneration of the axon extending to the solely at the suture lines.
first internode applies only to the most benign lesion: that of experi- Delay before repair leads to increasing fibrosis and to shrinking of
mentally crushing the nerve between the tips of a jeweller’s forceps. The the distal segment so that it becomes impossible to ensure an accurate
extent of longitudinal damage to a nerve is greater in a rupture caused topographical match.
by traction than it is in ‘tidy’ transections caused by knife or glass; this Destruction of the target tissues, of muscle and skin, limits function
effect worsens with increasing delay before repair, is worse still when even when there is strong regeneration.
healing has been complicated by sepsis, and worst of all in neglected Chronological age plays an important role: for example, signalling
ischaemia. mechanisms involved in the reinnervation of skeletal muscle may
Failure to repair main vessels and to ensure perfusion of tissues is become impaired with age (Kawabuchi et al 2011).
profoundly deleterious to regeneration and rules out worthwhile recov-
ery of function.
REFERENCES
Anand P, Birch R 2002 Restoration of sensory function and lack of long-term Li H, Terenghi G, Hall SM 1997 Effects of delayed re-innervation on the


chronic pain syndromes after brachial plexus injury in human neonates. expression of c-erbB receptors by chronically denervated rat Schwann
Brain 125:113–22. cells in vivo. Glia 20:333–47.
Anand U, Otto WR, Facer P et al 2008 TRPA1 receptor localisation in the Li H, Wigley C, Hall SM 1998 Chronically denervated rat Schwann cells
human peripheral nervous system and functional studies in cultured respond to GGF in vitro. Glia 24:290–303.
human and rat sensory neurons. Neurosci Lett 438:221–7. Lundborg G 2000 Brain plasticity and hand surgery: an overview. J Hand
Arthur-Farraj PJ, Latouche M, Wilton DK et al 2012 c-Jun reprograms Surg Br 25:242–52.
Schwann cells of injured nerves to generate a repair cell essential for Lundborg G 2003 Nerve injury and repair. A challenge to the plastic brain.
regeneration. Neuron 75:633–47. The Bunge Memorial Lecture. J Periph Nerv Syst 8:209–26.
Birch R, Misra P, Stewart MP et al 2012 Nerve injuries sustained during Mackinnon S, Dellon AL 1988 Surgery of the Peripheral Nerve. New York:
warfare: part II: Outcomes. J Bone Joint Surg Br 94:529–35. Thieme.
Birch R, St Clair Strange FG 1990 A new type of peripheral nerve lesion. Malessy MJ, Bakker D, Dekker AJ et al 2003 Functional magnetic resonance
J Bone Joint Surg 72B:312–13. imaging and control over the biceps muscle after intercostal-
Björkman A, Weibull A, Rosén B et al 2009 Rapid cortical reorganisation musculocutaneous nerve transfer. J Neurosurg 98:261–8.
and improved sensitivity of the hand following cutaneous anaesthesia Ochoa J, Fowler TJ, Gilliatt RW 1972 Anatomical changes in peripheral
of the forearm. Eur J Neurosci 29:837–44. nerves compressed by pneumatic tourniquet. J Anat 113:433–55.


Calenda G, Strong TD, Pavlovich CP et al 2012 Whole genome microarray Rabert D, Xiao Y, Yiangou Y et al 2004 Plasticity of gene expression in
of the major pelvic ganglion after cavernous nerve injury: new insights injured human dorsal root ganglia revealed by gene chip oligonucleo-
into molecular profile changes after nerve injury. BJU Int 109: tide microarrays. J Clin Neurosci 11:289–99.
1552–64.
Rosén B, Lundborg G 2007 Enhanced sensory recovery after median nerve
Carlstedt T 2007 Central Nerve Plexus Injury. London: Imperial College repair using cortical audio-tactile interaction. A randomised multicentre
Press. study. J Hand Surg Eur 32:31–7.
Christie KJ, Zochodne D 2013 Neuroscience Forefront Review. Peripheral Rosén B, Björkman A, Lundborg G 2011 Improving hand sensibility in vibra-
axon regrowth: new molecular approaches. Neuroscience 240:310–24. tion induced neuropathy: a case-series. J Occup Med Toxicol 6:13.
Dyck PJ, Nukada H, Lais CA et al 1984 Permanent axotomy: a model of Sai K, Kanamura A, Sibuya M et al 1996 Reconstruction of tonic vibration
chronic neuronal degeneration produced by axonal atrophy, myelin reflex in the biceps brachii reinnervated by transferred intercostal nerves
remodelling and regeneration. In: Dyck PJ, Thomas PK, Lambert EH et in patients with brachial plexus injury. Neurosci Lett 206:1–4.
al (eds) Peripheral Neuropathy, 2nd ed. Philadelphia: Elsevier, WB
Seddon HJ 1943 Three types of nerve injury. Brain 66:237–88.
Saunders, pp. 660–90.
Sulaiman OA, Gordon T 2009 Role of chronic Schwann cell denervation in
Dyck PJ, Dyck PJB, Engelstad J 2005 Pathological alterations of nerves. In:
poor functional recovery after nerve injuries and experimental strategies
Dyck PJ, Thomas PK (eds) Peripheral Neuropathy, 4th ed. Philadelphia:
to combat it. Neurosurgery 65:A105–14.


Elsevier, Saunders; Ch. 32, pp. 733–829.
Sunderland S 1951 A classification of peripheral nerve injuries producing
Gordon T 2015 The biology, limits, and promotion of peripheral nerve
loss of function. Brain 74:491–516.
regeneration in rats and humans. In: Tubbs RS et al (eds) Nerves and
Nerve Injuries. Elsevier, Academic Press; Ch. 61, pp. 993–1012. Suzuki H, Oyanagi K, Takahishi H et al 1993 A quantitative pathological
investigation of the cervical cord, roots and ganglia after long term ampu-
Hall S 2005 Mechanisms of repair after traumatic injury In: Dyck PJ, Thomas
tation of the unilateral upper arm. Acta Neuropathol (Berl) 85:666–73.
PK (eds) Peripheral Neuropathy, 4th ed. Philadelphia: Elsevier, Saun-
ders; Ch. 58, pp. 1403–34. Terenghi G, Calder JS, Birch R et al 1998 A morphological study of Schwann
cells and axonal regeneration in chronically transected human periph-
Htut M, Misra P, Anand P et al 2006 Pain phenomena and sensory recovery
eral nerve. J Hand Surg 23B:583–7.
following brachial plexus avulsion injury and surgical repair. J Hand
Surg 31B:596–605. Webber CA, Christie KJ, Cheng C et al 2011 Schwann cells direct peripheral
nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2.
Kawabuchi M, Tan H, Wang S 2011 Age affects reciprocal cellular interactions
Glia 59:1503–17.
in neuromuscular synapses following peripheral nerve injury. Ageing
Res Rev 10:43–53. Wright MC, Mi R, Connor E et al 2014 Novel roles for osteopontin and
clusterin in peripheral motor and sensory axon regeneration. J Neurosci
Knox ADC, Goswami R, Anastakis DJ et al 2015 Cortical plasticity after
34:1689–700.
peripheral nerve injury. In: Tubbs RS et al (eds) Nerves and Nerve Inju-


ries. Elsevier, Academic Press; Ch. 64, pp. 1055–71. Young JZ 1942 Functional repair of nervous tissue. Physiol Rev 22:318–74.
Lawson SN 2005 The peripheral sensory nervous system: dorsal root gan- Zigmond RE 2011 Gp130 cytokines are positive signals triggering changes
glion neurones. In: Dyck PJ, Thomas PK (eds) Peripheral Neuropathy in gene expression and axon outgrowth in peripheral neurons following
(2 vols), 4th ed. Philadelphia: Elsevier, Saunders; Ch. 8, pp. 163–202. injury. Front Mol Neurosci 4:62.
Lewis T, Pickering GW, Rothschild P 1931 Centripetal paralysis arising out
of arrested bloodflow to the limb. Heart 16:1–32.


CHAPTER
8
Preimplantation development
Understanding the spatial and temporal developmental processes that involve modifications of membrane sterols or surface proteins. They
take place within an embryo as it develops from a single cell into a traverse the cumulus oophorus and corona radiata, then bind to specific
recognizable human is the challenge of embryology. The control of glycoprotein receptors on the zona pellucida, ZP3 and ZP2. Interaction
these processes resides within the genome; fundamental questions of ZP3 with the sperm head induces the acrosome reaction, in which
remain concerning the genes and interactions involved in development. fusion of membranes on the sperm head releases enzymes, such as
acrosin, which help to digest the zona around the sperm head, allowing
the sperm to reach the perivitelline space. In the perivitelline space,
STAGING OF EMBRYOS
the spermatozoon fuses with the oocyte microvilli, possibly via two
disintegrin peptides in the sperm head and integrin in the oolemma
For the purposes of embryological study, prenatal life is divided into (Figs 8.4, 8.5A).
an embryonic period and a fetal period. The embryonic period covers Fusion of the sperm with the oolemma causes a weak membrane
the first 8 weeks of development (weeks following ovulation and fertil- depolarization and leads to a calcium wave, which is triggered by the
ization resulting in pregnancy). The ages of early human embryos have sperm at the site of fusion and crosses the egg within 5–20 seconds. The
previously been estimated by comparing their development with that calcium wave amplifies the local signal at the site of sperm–oocyte
of monkey embryos of known postovulatory ages. Because embryos interaction and distributes it throughout the oocyte cytoplasm. The
develop at different rates and attain different final weights and sizes, a increase in calcium concentration is the signal that causes the oocyte to
classification of human embryos into 23 stages occurring during the resume cell division, initiating the completion of meiosis II and setting
first 8 weeks after ovulation was developed most successfully by Streeter off the developmental programme that leads to embryogenesis. The
(1942), and the task was continued by O’Rahilly and Müller (1987). pulses of intracellular calcium that occur every few minutes for the first


An embryo was initially staged by comparing its development with that few hours of development also trigger the fusion of cortical granules
of other embryos. On the basis of correlating particular maternal men- with the oolemma. The cortical secretory granules release an enzyme
strual histories and the known developmental ages of monkey embryos, that hydrolyses the ZP3 receptor on the zona pellucida and so prevents
growth tables were constructed so that the size of an embryo (specifi- other sperm from binding and undergoing the acrosome reaction, thus
cally, the greatest length) could be used to predict its presumed age in establishing the block to polyspermy. The same cortical granule secre-
postovulatory days (synonymous to postfertilizational days). O’Rahilly tion may also modify the vitelline layer and oolemma, making them
and Müller (2000, 2010) emphasize that the stages are based on external less susceptible to sperm–oocyte fusion and providing a further level of
and internal morphological criteria and are not founded on length or polyspermy block.
age. Ultrasonic examination of embryos in vivo has necessitated the The sperm head undergoes its protamine → histone transition as the
revision of some of the ages related to stages, and embryos of stages second polar body is extruded. The two pronuclei grow, move together
6–16 are now thought to be up to 3 to 5 days older than the previously and condense in preparation for syngamy and cleavage after approxi-
used embryological estimates (O’Rahilly and Müller 1999, 2010). mately 24 hours (Fig. 8.5B). Nucleolar ribosomal ribonucleic acid
Within this staging system, embryonic life commences with fertilization (rRNA), and perhaps some messenger RNA (mRNA), are synthesized
at stage 1; stage 2 encompasses embryos from two cells, through com- in pronuclei. A succeeding series of cleavage divisions produces eight
paction and early segregation, to the appearance of the blastocele. The even-sized blastomeres at approximately 55 hours, when embryonic
developmental processes occurring during the first 10 stages of embry- mRNA is transcribed.
onic life are shown in Figure 8.1. Several examples of cells that contain male and female pronuclei,


Much of our knowledge of the early developmental processes is termed ootids, have been described. Pronuclear fusion as such does not
derived from experimental studies on amniote embryos, particularly the occur; the two pronuclear envelopes disappear and the two chromo-
chick, mouse and rat. Figure 8.2 shows the comparative timescales of some groups move together to assume positions on the first cleavage
development of these species and human development up to stage 12. spindle. No true zygote containing a membrane-bound nucleus is
The size and age, in postovulatory days, of human development from formed.
stage 10 to stage 23 is given in Figure 8.3. The presence of the pronuclei from both parental origins is crucial
Information on developmental age after stage 23 (8 weeks post for spatial organization and the controlled growth of cells, tissues and
ovulation) is shown in Figure 14.3, where the developmental staging organs. In the mouse, embryos in which the paternal pronucleus has
used throughout this text is juxtaposed with the obstetric estimation of been removed and replaced with a second maternal pronucleus develop
gestation that is used clinically. A critique of staging terminology and to a relatively advanced state (25 somites), but with limited develop-
the hazards of the concurrent use of gestational age and embryonic age ment of the trophoblast and extraembryonic tissues. In contrast,
is given in Chapter 14; sizes and ages of fetuses towards the end of embryos in which the maternal pronucleus has been replaced by a
gestation are illustrated in Fig. 14.9. second paternal pronucleus develop very poorly, forming embryos of
only six to eight somites, but with extensive trophoblast. Thus it seems
that the maternal genome is relatively more important for the develop-
FERTILIZATION
ment of the embryo, whereas the paternal genome is essential for the
development of the extraembryonic tissues that would lead to placental
The central feature of reproduction is the fusion of the two gamete formation.
pronuclei at fertilization. In humans, the male gametes are spermato- This functional inequivalence of homologous parental chromo-
zoa, which are produced from puberty onwards. Female gametes are somes is called parental imprinting. The process causes the expression
released as secondary oocytes in the second meiotic metaphase, usually of particular genes to be dependent on their parental origin; some genes


singly, in a cyclical fashion. The signal for the completion of the second are expressed only from the maternally inherited chromosome and
meiotic division is fertilization, which stimulates the cell division cycle others from the paternally inherited chromosome. The genes involved
to resume, completing meiosis and extruding the second polar body are called imprinted genes. The requirement for both parental genomes
(the second set of redundant meiotic chromosomes). is limited to a subset of the chromosomes. Uniparental disomy can arise
Fertilization normally occurs in the ampullary region of the uterine through meiotic and mitotic non-disjunction events, and results in
tube, probably within 24 hours of ovulation. Very few spermatozoa individuals who are completely disomic or who exhibit mosaicism of
reach the ampulla to achieve fertilization. They must undergo capacita- disomic and non-disomic cells. If imprinted genes reside on the affected
tion, a process that is still incompletely understood, and which may chromosomes, then the uniparental disomic cells will either express a 163


PreimPlantation develoPment
164
2
noitCeS
Syncytio- Lacunae Intervillous
Tropho- trophoblast spaces
Oocyte blast Cyto-
Chorion Villi
trophoblast
Ootid
Visceral Extraemb. mesenchyme Haemopoiesis
hypoblast
Hypoblast Primary yolk Secondary
Zygote Parietal sac yolk sac
hypoblast
Inner Allantois
cell
mass
Extraemb.
Connecting stalk
mesoblast
Epiblast
Amnion
Primordial germ cells
Primitive streak
Notochordal
process/plate
Embryonic
endoderm
Mesoblast Mesenchyme
Somatopleuric and
splanchnopleuric coelomic epithelium
Epithelial somites
Caudal eminence
Neural
ectoderm
Neural
crest
Ectodermal placodes
Surface
ectoderm
Fig . 8 .1 Developmental processes occurring during the first 10 stages of development . In the early stages, a series of binary choices determine the cell
lineages . Generally, the earliest stages are concerned with formation of the extraembryonic tissues, whereas the later stages are concerned with the
formation of embryonic tissues .
double dose of the gene or have both copies repressed. For example, tation genetic diagnosis (PGD). A sample (biopsy) is removed from
the gene encoding the embryonal mitogen insulin-like growth factor II either the oocyte (polar bodies), the embryo itself (a blastomere) or the
is expressed from the paternally inherited chromosome, and repressed blastocyst (small piece of trophectoderm), and subjected to specific
when maternally inherited. genetic testing. Oocytes and embryos can also be biopsied and screened
for aneuploidy (errors in chromosome copy numbers) in preimplanta-
In vitro fertilization tion genetic screening (PGS). Unaffected embryos can then be identified
for transfer to the patient. Embryos that are surplus to immediate thera-
peutic requirements can also be cryopreserved in liquid nitrogen for
In vitro fertilization (IVF) of human gametes is a successful way of
later use. Propanediol or dimethylsulphoxide are commonly used as


overcoming most forms of infertility (Video 8.1). Controlled stimula-
cryoprotectants for early embryos, and, like glycerol, may be used for
tion of the ovaries (e.g. pituitary downregulation using gonadotrophin-
blastocysts. Conception rates per cycle using ovarian stimulation, IVF
releasing hormone superactive analogues, followed by stimulation
and successive transfers of fresh and cryopreserved embryos exceed
with purified follicle stimulating hormone or urinary menopausal
those obtained during non-assisted, or natural, conception.
gonadotrophins) enables many preovulatory oocytes (often 10 or
more) to be recruited and matured, and then aspirated transvaginally
using ultrasound guidance, 34–38 hours after injection of human cho- PREIMPLANTATION DEVELOPMENT
rionic gonadotrophin (which is given to mimic the luteinizing
hormone surge). These oocytes are then incubated overnight with
Cleavage
motile spermatozoa in a specially formulated culture medium, in an
attempt to achieve successful in vitro fertilization. In cases of severe
male-factor infertility, in which there are insufficient normal spermato- The first divisions of the zygote are termed cleavages. They distribute
zoa to achieve in vitro fertilization, individual spermatozoa can be the cytoplasm approximately equally among daughter blastomeres, so,
directly injected into the oocyte in a process known as intracytoplas- although the cell number of the preimplantation embryo increases, its
mic sperm injection (ICSI), which is as successful as routine IVF. In total mass actually decreases slightly (Fig. 8.6). Cell division can be
cases in which there are no spermatozoa in the ejaculate, suitable asynchronous and daughter cells may retain a cytoplasmic link through
material can sometimes be directly aspirated from the epididymis or much of the immediately subsequent cell cycle via a midbody, as a
surgically retrieved from the testes, and the extracted sperm are then result of the delayed completion of cytokinesis. No centrioles are
used for intracytoplasmic injection of sperm. present until 16–32 cells are seen, but amorphous pericentriolar mat-


It is also now possible, in some cases, to test embryos for the pres- erial is present and serves to organize the mitotic spindles, which are
ence of a particular genetic or chromosomal abnormality by preimplan- characteristically more barrel- than spindle-shaped at this time.
egats
fo
noitpircseD
noitazilitreF
1
egavaelc
tsriF
noitatnalpmierP
2
noitcapmoC tsycotsalb
eerF
3
anoz
morf
dehctah
4
noitatnalpmI
a5
detnalpmI
b5
sulliverp
c5 a6
cas
kloy yradnoceS
b6
kaerts
evitimirP
7
drohcotoN lanac
ciretnerueN
8
gnidlof
erofeB
evoorg
larueN
9
setimoS moleoc
.bmeartnI
ebut
larueN
01
gnidlof
retfA
tserc
larueN
11
Approx. 1 2–3 4–5 6 16–18 18–21 24–27 28–30
age in days 7–12 21–25 26–29