Multiple sclerosis (MS) is a multifocal Demyelinating disease of
the central nervous system (CNS) which can lead to sever physical and cognitive
disability and neurological defects (1). Damage to this myelin sheath protecting the nerve cells in the
brain and spinal cord progresses to damage or destruction of the axons (nerve
fibers) over time leading to irreversible neurodegeneration explaining the
progression of the disease and the increase in disability (2).
Unfortunately, there is no cure for MS and current remedies only
help in alleviating the symptoms and halting the immune attack (3). But Stem cell therapies offers a new hope for treatment of such
neurological diseases, as stem cells was efficiently proven to differentiate
effectively into oligodendrocytes and astrocytes in vitro and in vivo (4) and secretes neurotrophic factors having immunomodulatory effects
preventing further damage and creating a regenerative microenvironment for
remyelination (5). Based on these results, several small pilot clinical trials in
subjects with advanced MS have demonstrated that Mesenchymal Stem Cells (MSCs)
administration is safe and provided an early signal of clinical effectiveness (6).
Adipose Derived Stem Cells (ADSCs) is a population of MSCs which
have a much higher frequency in the adipose tissue than in bone marrow
(approximately 500-fold more). Moreover, ADSCs can be harvested by minimally
invasive procedures that should facilitate their use in cell transplantation
(Tsuji, Rubin, & Marra, 2014). These cells are capable to differentiate to
other cells outside their lineage, such as neural progenitors and
oligodendrocytes. Moreover, ADSCs through paracrine effects are able to promote
survival and proliferation of endogenous oligodendrocyte precursor cells which
leading to further the process of remyelination. Another relevant finding of
previous study was that a high percentage of ADSCs which transplant in rat
model of MS, expressed Olig2 and MBP that are special markers of oligodendrocyte
(7,8). Therefore, ADSCs transplantation can induce nerve repair and
provide a practical way for remyelination in neurodegenerative diseases.
In order to maximize the cellular proliferation efficiency of
ADSCs, low level laser irradiation was used to activate the cells. Laser
irradiation at different intensities has been recognized to inhibit and/or
stimulate cellular processes. Recent findings suggest that at the cellular
level, laser energy of a particular wavelength can initiate signaling cascades,
such as those that promote cellular proliferation (Mvula, Moore, &
While administration of ex vivo culture-expanded stem cells has
been used to study immunosuppressive mechanisms in multiple models of
autoimmune diseases, less is known about the uncultured, nonexpanded stromal
vascular fraction (SVF)-based therapy. The SVF is composed of a heterogeneous
population of cells and has been used clinically to treat acute and chronic
diseases, alleviating symptoms in a range of tissues and organs (Blaber et al.,
2012). Equine and canine studies demonstrating anti-inflammatory and
regenerative effects of non-expanded SVF cells have yielded promising results
(Abdallah, Shamaa, El-Tookhy, & Abd El-Mottaleb, 2015).
the ability of human SVF cells was compared with culture expanded
ADSCs and bone-derived marrow stromal cells (BMSCs) as a treatment of myelin
oligodendrocyte glycoprotein (35–55)-induced experimental autoimmune
encephalitis in mice, a model of MS. The data indicated that intraperitoneal
administration of all cell types significantly ameliorates the severity of
disease. Furthermore, the data also demonstrated, for the first time, that the
SVF was as effective as the more commonly cultured BMSCs and ADSCs in an MS
The aim of this
work is the histological evaluation of the transplantation of non-expanded
Laser activated Adipose derived SVF in a Dog Model of Toxin induced MS.
All animal’s experimental protocols
were approved by the Institutional Animal Care and Use Committee (IACUC) of
Faculty of Veterinary Medicine, Cairo University. Approval ID#: CU/II/S/23/16.
dogs (2-5 years of age and of both sexes) were used in this study. All dogs
will be subjected to a pre-study evaluation excluding any animals suffering
from any nervous manifestations as paralysis, paraplegia, tremors, paresis,
lameness, head tilts, etc…
were equally and randomly allocated in 2 main groups:
(Control group) Induction of the MS using Gliotoxin Ethidium bromide and
treatment using Normal Saline at day 14 post induction.
(Treated group) Induction of MS and Treatment using Stem cells at day 14 post
group was then subdivided into 4 equal subgroups according to the period of
observation 3,7, 14, 28 days’ post treatment.
of Demyelinating lesions: Under general anesthesia, bilateral
holes were drilled using a dental drill in the dorsal lamina of the 1st
lumbar vertebra. An amount of 20 µl
of 0.1 % Ethidium bromide was injected in the lateral columns of the spinal
cord using a microneedle syringe attached to a capillary tube (10).
Animals were sacrificed at days 3, 7, 14 and 28 post injection, and
coronal sections of 1mm of the spinal cord were taken for histopathology and
immunohistochemistry were fixed on neutral buffered formalin (11,12).
cell isolation and preparation:
tissue collection: Under general anesthesia, a small skin incision (2-3 cm) was
aseptically operated and adipose tissue (15-20 g) was collected from
sub-cutaneous fat in the abdomen into a 50 mL sterile cup.
processing and isolation of SVF: The collected fat pad was minced and
washed extensively with phosphate-buffered saline and then an equal volume of
0.1% collagenase type 1 (Sigma, Aldrich) was added. The tissue was placed in a
rotary incubator at 37°C, with continuous agitation for 1 h. After digestion,
the lipoaspirates were centrifuged at 1200 rpm for 10 min to separate the
lipoaspirate and the collagenase. The lipoaspirates were then rewashed 3 times
to remove any remaining collagenase. After the last round of centrifugation,
cells in the aspirates were counted using a hemocytometer and the viability of
the cells was assessed using the trypan blue dye exclusion test.
level laser activation: The prepared cells seeded in the PRP
were exposed to low level laser for 20 min using LED technology (Adilight 2®
Australia) at 3 laser diodes for 3 frequencies one in the red, one in the green
and one in the yellow.
4. Stem cells labeling: Labeling of the stem cells with
PKH26 stain was carried out according to manufacture guidelines
(Sigma-Aldrich). Briefly, SVF cell suspension was prepared by adding 1 mL of
diluents and incubated the cell/dye suspension for 1–5 min. The staining was
stopped by adding an equal volume of cold 1 % bovine serum albumin to the cell
mixture, and labeled cells were washed twice with complete medium. After PKH-26
staining, an aliquot of cells was assessed by fluorescent microscopy to
determine the staining efficiency.
of SVF: The stem cell preparation (10 X106 nucleated cells) in
normal saline was injected directly in the Cerebrospinal fluid (CSF) through
the foramen magnum in the proximal spinal cistern at the day 14 from the
induction of the MS as mentioned by (13).
Evaluation of the treatment:
6.1.Histopathological evaluation:The tissue samples
were embedded in paraffin blocks, were sectioned into 10-µm-thick sections.
sections were brought to distilled water then Stained with the alum
haematoxylin for 5 mins and rinsed in running tap water. Stain with eosin for 2
mins then dehydrated, cleared and mounted. And sections were examined using
6.2.Immunohistochemistry:Antibodies used for
immunohistochemistry were Anti-Bax antibody E63 (ab32503, Abcam®),
Anti-Caspase-9 antibody (ab52298 Abcam®), Anti-GFAP antibody
(ab7260, Abcam®), Anti-Myelin Basic Protein antibody (ab40390, Abcam®), Anti-Olig2 antibody (ab136253, Abcam®)
with hematoxylin as counter stain.All sections
were deparaffinized, and antigen retrieval was performed in 0.01 Km Tris- EDTA
(Sigma-Aldrich; pH 9.0) in a benchtop autoclave for 5 minutes. Sections were
pretreated with 10% fetal calf serum (Invitrogen, Dublin, Ireland)/TBS (Sigma-
Aldrich) for 20 minutes at room temperature. All antibodies were then incubated
on sections overnight at 4-C and detected using peroxidase-labeled anti-rabbit
secondary antibodies (Dako) with diaminobenzidine (Dako) as chromogen (15).