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\begin{document}

\title{Draft 2}



\author[1]{safwana}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 05, 2024}


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The limitation of light microscopy in the study of mast cells prompted
the employment of other imaging platforms. A platform that was widely
used in the early sixties through mid-eighties was electron microscopy.
Using the RCA EMU-2A electron microscope,~\citet{Smith_1957}~studied
mast cells isolated -- via centrifugation -- from the peritoneal fluid
of adult male rodents. The cells were dehydrated with various alcohol
concentrations before they were embedded in plastic and gelatin and cut
into thin sections prior to imaging. The authors noted the subpar
quality of the electron micrographs in preserving the structural
integrity of the mast cells.~However, the authors stressed that~the mast
cells that they obtained appeared similar to those isolated from skin,
spleen, and liver that were prepared according to the method developed
by~\cite{14927794}.~~ The morphology of normal mast cells was
described as having a large nucleus occupied by thread-like structures
with empty spaces towards the interior, multiple round or oval-like
electron-dense bodies with a diameter of 0.5-1.0 \selectlanguage{greek}µ \selectlanguage{english}(Smith 1957).
Thread-like materials that resemble those in the nucleus were present
inside these electron-dense structures (cytoplasmic granules), albeit
they were thinner with fine granule-like~particles attached to them.~ In
addition to imaging normal mast cells, the effects of total body
irradiation and intraperitoneal (IP) injection of heparin-binding agent
-- toluidine blue and protamine sulfate -- and histamine-releasing
agent, stilbamidine~were investigated. The size of the granules and the
inner structures remained unchanged in mast cells isolated from
IP-injected rats. However, each granule seemed to be surrounded by a
halo of clear cytoplasm enclosed and separated from each other by
the~perigranular membrane. Unlike the untreated and IP-injected mast
cells, cells isolated from irradiated rodents had more electron-dense
bodies that were much larger than the typical size range of a secretory
granule with fewer inner reticular structures. The nucleus appeared
elongated and deformed and together with the lack of intragranular
structures, mirrored that observed under the light microscope, which is
suggestive of cells undergoing
apoptosis~\protect\hyperlink{Smith_1957}{\emph{(Smith, 1957)}}.~~

The plasma membrane of rat peritoneal mast cells was reported to exhibit
finger-like projections that protrude into the extracellular
space~~\protect\hyperlink{Lagunoff_1972}{\emph{(Lagunoff, 1972)}}. This
structural component was also observed in rat mast cells in the
tongue~\protect\hyperlink{Enerb_ck_1974}{\emph{(Enerbck et al.,
1974)}}. However, this~microvillus-like folding of the membrane was
absent in mast cells observed in the lamina propria of the gut mucosa.
In addition to the smooth cell membrane, mucosal mast cells often
exhibited irregularly-shaped nuclei with
indentations~\emph{\protect\hyperlink{Enerb_ck_1974}{(Enerbck et al.,
1974)}~}with\emph{~}fewer but larger granules as compared to tongue mast
cells, which contained numerous and fairly-uniform sized granules that
were within the size range reported by Smith (1957). The intragranular
structures in tongue mast cells exhibited a homogeneous fine particulate
appearance whereas those in mucosal mast cells were composed of coarse
particles. Besides this, other mesenchymal and granular (mainly
eosinophils) cell types as well as phagocytic immune cells, were
typically found in contact or in close proximity to mucosal mast
cells~~\protect\hyperlink{Enerb_ck_1974}{\emph{(Enerbck et al.,
1974)}}. They also appeared smaller than peritoneal mast cells and were
often found localised near nerves and blood
vessels~~\protect\hyperlink{Lagunoff_1972}{\emph{(Lagunoff, 1972)}}.

~The morphology of human mast cells under normal/untreated conditions
was explored using electron microscopy. Tissue biopsies were obtained
from digital and abdominal skin and subcutis, as well as the gastric
mucosa ~\protect\hyperlink{Hibbs_1960}{\emph{Hibbs et al. (1960)}}. The
samples were divided for imaging by light and electron microscopy where
the tissue specimens were processed as described by Smith (1957). Two
morphologically distinct cells were observed: One was spindle-shaped
with an elongated nucleus and numerous tightly-packed granules wherein
the internal structures were not visually apparent; the other type was
round or ovoid with many cytoplasmic granules that were more widely
distributed. Unlike the light microscope, at the highest magnification
of X50 000 of the objective lens of the electron microscope, the authors
were able to observe the intragranular structures in the ovoid cells.
They appeared to consist of two components -- fine particles and
lamellar/scroll-like structures. Variations exist between cells with
regard to the composition and localisation of these components such that
the granules may be composed of mostly fine particles or lamellar or a
mix of both. Similar to the skin and dermis, both cell types were
observed in the gastric mucosa specimen. However, only the ovoid cells
were found in the specimen obtained from a patient who received
intensive adrenocorticosteroid treatment. Furthermore, only the lamellar
structure within the granules could be observed; the other component was
rarely found in these cells, which lead the authors to the hypothesis
that the steroid treatment caused the fine particles to be released from
the granules. The study also highlighted another advantage of electron
microscopy -- it enabled the identification of two cell types as
distinct cells, which was not feasible via the light
microscope~~\protect\hyperlink{Hibbs_1960}{\emph{(Hibbs et al., 1960)}}.

Electron microscopic studies of mast cells have been done with
ultra-thin sections of tissue specimens and liquid samples from various
tissues and mammals.~~\protect\hyperlink{Smith_2006}{\emph{Smith
(2006)}}~compiled a list of studies highlighting the source of mast
cells that were utilised to-date and noted that the cells had undergone
similar processes prior to imaging, that is, the cells were fixed with
osmium tetroxide and methacrylate, followed by embedding in plastic/Epon
resin, and stained with uranyl acetate. Micrographs from these
individual studies of normal mast cells appeared similar across species
(Smith 2006). The separation between granules by a two-layer
electron-dense structure was evident in untreated mast cells, although
in certain cells, many granules were was only partially surrounded by
this~perigranular membrane
(\protect\hyperlink{Lagunoff_1972}{\emph{Lagunoff 1972)}}. The
variability in the intragranular structures observed by Hibbs et al.
(1960) was evident~across different mast cell types of different mammal
species.

Degranulation Processes via Electron Microscopy

~

Changes in mast cells with regard to their structure and morphology
during degranulation have been studied with several histamine-releasing
agents (Bloom 1967, Lagunoff 1973). Compared with untreated cells where
all of the granules appear homogenously electron-dense, treated cells
were composed of both unaltered and altered granules where the latter
appeared to be less dense and separated from their perigranular
membrane. The inner structures of these granules appeared as filaments
composed of fine particles, leading to the reduction in electron density
(Lagunoff 1973).~ These less-dense granules appeared to localise near
the periphery of the cell (although many inner granules were also
involved in the degranulation process) and enclosed in larger vacuoles
of cytoplasmic matrix that open up to the outside of the
cell~\protect\hyperlink{Bloom_1967}{\emph{(Bloom 1967)}}. However, this
extracellular opening was not detected in mast cells treated with
polymyxin B~sulfate~(Lagunoff, 1973). Altered granules were detected
extracellularly, indicative of the release of granular contents
(histamine) upon stimulation.~ An electron-lucent space in between the
granules and the perigranular membrane was also evident, mirroring the
observation by Smith (1957). At high magnification, adjacent
perigranular membranes came into contact with each other forming a
five-layer membrane~\protect\hyperlink{Lagunoff_1973}{\emph{(Lagunoff,
1973)}} and the resulting close proximity in the intergranular space was
suggested to result in the formation of the extensive cytoplasmic
channel along the cell periphery (Lagunoff 1972). Perigranular membrane
at the periphery of mast cells also fused with the plasma membrane; in
several micrographs, the fused membrane was connected to an
extracellular protrusion that resembled an early pore formation, which
might be involved in histamine release that differs from the exocytosis
model ~\protect\hyperlink{Lagunoff_1973}{\emph{(Lagunoff, 1973)}}.~ ~ ~~

The degranulation response varies across mast cell types and the
histamine-releasing agents. Certain chemical mediators -- polymyxin B
sulfate, compound 48/80, bee venom -- produce similar morphological
changes~\cite{4179625}~\citep{Bloom_1967}\citep{4158508} in the
skin, subcutis, and peritoneal mast cells. Other chemicals such as
n-decylamine resulted in a more extensive change in mast
cells~~\protect\hyperlink{Bloom_1967}{\emph{(Bloom 1967)}}. Both plasma
and perigranular membranes disintegrated and the less dense granules
were randomly distributed among electron-dense granules throughout the
cytoplasm. At the opposite end of this spectrum, gut mucosal mast cells,
which exhibited the aforementioned morphological differences as compared
to untreated peritoneal mast cells, were unaffected when treated with
compound 48/80, suggesting functional differences exist between mast
cells isolated from different sites.~ ~ ~~

In addition to transmission/scanning electron microscopy,
freeze-fracture electron microscopy offered a different outlook on mast
cell degranulation that complimented the micrographs of stimulated mast
cells~\citep{Chi_1976}. In unstimulated cells, the granules appeared
as bulges with evenly distributed particles within the boundary of the
bulges (intramembrane particles). In cells incubated with polymyxin B,
there were patches in the bulges where the intramembranous particles
were absent. These particles appeared to be concentrated at the base of
the bulges instead. A pore was also observed that opened into a large
channel corresponding to the fusion of vacuoles in mast cells that were
fixed at a later time-point after stimulation. Intramembranous particles
were detected near the lining of the membrane of the channel, indicative
of granular release \citep{Chi_1976}.~

Corroborative findings regarding unstimulated and stimulated mast cells
facilitated investigative studies on factors that may induce mast cell
degranulation. A clinical study by~\citet{Casale_1984} ~investigated the
applicability of opiates to be used as a routine test for dermal
hypersensitivity in the clinic. Electron micrographs of mast cells
obtained from healthy subjects that were injected intradermally with
endogenous opioids and opiates were assessed in addition to the
wheal-and-flare skin test. Positive results from this test -- the
formation of wheals and flares -- correlated with features of
degranulated mast cells as established in literature; this indicated
that mast cells could be stimulated by opiates and endogenous opioids
through the engagement of opiate receptors. Another study showed the
role of skin mast cells in neuroinflammatory skin diseases such as
psoriasis and atopic dermatitis where the conditions are induced or
worsened by stress~\cite{10469524}. A mouse model of acute
immobilisation was used to trigger the release of corticotropin
releasing hormone (CRH)~and neurotensin. Mast cells from the stressed
animal exhibited the characteristics of degranulation, which were not
apparent when the animals were pre-treated with anti-CRH and neurotensin
receptor antagonist. Results from the micrographs augmented the other
experimental read-outs, indicating that mast cell degranulation may be
implicated in human neurogenic skin disorders (Singh 1999).~

Segregation of Lyn from the Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β \selectlanguage{english}clusters was observed in activated
cells while Syk appeared to colocalise with the Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β

\selectlanguage{english}on These anti-DNP IgE-primed cells exhibited clusters of Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β
\selectlanguage{english}composed of more than 10 particles when activated with DNP-BSA, which
was larger than the 2-3 particle clusters that were mostly observed in
resting cells. The distribution of Lyn was similar in resting cells and
these clusters often localised with Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β. \selectlanguage{english}In contrast, Syk formed
individual clusters or appeared as singlets, separated from the Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β
\selectlanguage{english}clusters. In activated mast cells, the co-localisation of Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β \selectlanguage{english}and
Syk gold particles was observed, whereas Lyn appeared to aggregate at
the periphery of these Syk-Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}β \selectlanguage{english}clusters, forming a gold string. An
insight into the membrane topography and behaviour of the IgE receptor
and its associated proteins upon activation was achieved via high
resolution TEM. {}







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