Red to yellow bone marrow distribution in the mouse skeleton in

Patrick Helbling,
Timo Geitlinger
Red to yellow bone marrow distribution in the mouse skeleton in
homeostasis and radiation-induced marrow failure.
Timo Geitlinger, Kantonsschule Zürich Nord
Patrick Helbling, Gymnasium Oberwil
Supervision: Evangelos Panopoulos, Josefine Tratwal, EPFL
Introduction
Bones not only serve as mechanical support, protection, mobility and mineral storage
but also as center of blood production (hematopoiesis). Hematopoiesis takes place in the
hematopoietic (red) bone marrow (reference 4). There is also another type of bone marrow in
the skeleton, which is adipocytic (yellow) that has been shown to be inhibitory of
hematopoiesis (reference 5).
The therapy of patients with leukemia (blood cancer) aims to replace the sick bone
marrow with new cells by stem cell transplantation. Following the elimination of the bone
marrow the bones fill with adipocytes, before the patients have again a functional blood
system with hematopoietic (red) marrow. During this time, patients are prone to lifethreatening infections and bleeding.
Since mouse models are close to human as far as evolution is concerned, they serve to
study these processes. Using them, we can investigate the bone marrow functions in the
healthy individual and in disease (e.g. leukemia), test and improve methods of therapy
(reference1).
Material and Methods
Female C57/Bl6 mice (figure 1) 8 to 12 weeks old, were used as source of tissues.
Bones were sampled and cleaned from muscles and tendons (figure 2). Then they were fixed
in 10% v/v formalin for 24 hours. During the next 36 hours they were decalcified in a solution
of tri-sodium citrate 20% v/v and formic acid 98% v/v.
The bones were later dehydrated overnight in baths of increasing ethanol
concentration and then successive xylene baths. Finally they were infiltrated by paraffin,
embedded in paraffin blocks, cut in 5µm sections, and stained with hematoxylin and eosin
(H&E). The histological sections (figure 3) were photographed with an Olympus© VS120
slide scanner. Photos were extracted and quantified with the ImageJ software.
Figure 1: a C57/Bl6 mouse
Figure 2: Bones cleaned
from muscles and tendons
Figure 3 :H&E staining of a femur
section in photonic transmission
microscopy (magnification x20)
Patrick Helbling,
Timo Geitlinger
Red and Yellow Bone Marrow Distribution in Homeostasis
As it is visible in figure 4, in a homeostatic adult mouse, the quantity of the red bone
marrow is much bigger in the forward region of the spine than in the rear region. The lumbar
(L) vertebrae have mostly red bone marrow whereas the caudal vertebrae (Cd) are mostly
yellow.
tail
head
Figure 4: H&E staining of a sagittal section of an adult mouse spine in homeostasis
(magnification x20)
T: vertebrae thoracicae
S: vertebrae sacrales
L: vertebrae lumbales
Cd: vertebrae Caudales
At birth, all of the bone marrow is red and hematopoietic, capable to produce all blood
cells. The red marrow is gradually replaced by adipocytic marrow with age. In humans this
process takes place up to the approximate age of 25 years. For the rest of the lifetime the red
bone marrow is mainly located in the central skeleton (reference 4). Even if the red to yellow
conversion slows down with age it can be influenced by external factors. As indicated in
bibliography (reference 5) low temperature and stress can have a positive impact on the
conversion.
Clinical relevance of the model: Radiation-induced bone marrow failure
and reconstitution
In case of a disease like leukemia, which originates in the bone marrow, it is important
to replace the ill marrow with cells from a healthy donor. In the Laboratory of Regenerative
Haematopoiesis, mouse models are used to simulate, observe and improve this process.
To make this transplantation possible, the entire body is irradiated in order to destroy
the bone marrow. Bone marrow from a mouse with identical genetic background (syngenic) is
then injected in the tail vein.
Figure 5: Irradiation and bone marrow transplantation
Patrick Helbling,
Timo Geitlinger
By irradiation, the marrow environment is made available for the graft while the immune
system becomes suppressed. During this process the red marrow of the recipient mouse
converts to yellow marrow. Progressively the bone marrow from the donor starts to engraft,
the bone marrow becomes redder and the immune system is re-established (figures 6 and 7)
Figure 6: Evolution of the femur bone marrow histology during the experiment. Transplantation
in day 0, replacement of the red bone marrow by yellow (days 5 to 20) and engraftment of the
red bone marrow 25 days after the transplantation. (H&E staining, magnification x20)
% of bone marrow
50 40 30 Yellow
20 Red
10 0 0 5 10 15 20 25 days post-transplant
Figure 7: Quantification of bone marrow types (red vs yellow) during the experiment.
New treatments are being tested to shorten this period of immune suppression, that
correlates with the period when there is only very little heamapoietic bone marrow and mainly
yellow marrow present in the bones. A shortening of this period is important to decrease the
possibility of fatal infections in humans and in the mouse model.
Patrick Helbling,
Timo Geitlinger
Conclusion
The red and yellow marrows are two major components of the bone marrow. Their
environment also consists of other cell types that add more levels of complexity by giving
signals and modifying the surrounding space (reference 2). Study of the interactions between
the cells will improve our knowledge of the natural state in the bone marrow and how to
improve treatments. This, together with closer study in the hematopoietic bone marrow cell
populations will help us to propose more refined transplantation methods.
Acknowledgements
First of all we want to thank the organization “Schweizer Jugend Forscht” for enabling
and planning this great week full of great experiences.
We would like to thank Professor Naveiras for giving us the opportunity to evolve in a
very enriching and stimulating environment. Thanks also to Mr. Vasco Campos for sharing
his data with us. Special thanks to Ms. Josefine Tratwal and Mr. Evangelos Panopoulos for
teaching, supporting and opening a completely new point of view on sciences.
Bibliographic references
1. Bianco P, “Mesenchymal” Stem Cells, Annu Rev Cell Dev Biol. 2014;30:677-704
2. Frenette PS, Pinho S, Lucas D, Scheiermann C., Mesenchymal stem cell: keystone of
the hematopoietic stem cell niche and a stepping-stone for regenerative medicine.,
Annu Rev Immunol. 2013;31:285-316.
3. Komàrek V, Sidlistni, Lysolaje, Mouse Gross Anatomy, The Laboratory Mouse,
Elsevier Ltd, 2004.
4. Kricun ME, M.D., Red-yellow marrow conversion: Its effect on the location of some
solitary bone lesions, Skeletal Radiol, 1985, 14:10-19
5. Naveiras O, Nardi V, Wenzel PL, Hauschka PV, Fahey F, Daley GQ, Bone-marrow
adipocytes as negative regulators of the haematopoietic microenvironment, Nature.
2009 Jul 9;460(7252):259-63