Influence of spinal cord protein hydrolysate upon the

ORIGINAL ARTICLE
Folia Neuropathol.
Vol. 41, No. 1, pp. 29–34
Copyright © 2003 Via Medica
ISSN 1641–4640
Influence of spinal cord protein hydrolysate upon
the blood brain barrier changes due to experimental
allergic encephalomyelitis in Lewis rats.
Ultrastructural study
Barbara Kwiatkowska-Patzer1, Bożena Baranowska3, Michał Walski2, Andrzej W. Lipkowski1
1 Neuropeptide
2 Laboratory
Laboratory, Medical Research Centre, Polish Academy of Sciences, Warszawa, Poland
of Cell Ultrastructure, Medical Research Centre, Polish Academy of Sciences, Warszawa, Poland
3 Industrial Chemistry Research Institute, Warszawa, Poland
A specific protein (antigen) given orally is a known method of introducing tolerance of immunological response to this antigen. This method has recently been reviewed by some authors as a possible tool in the
treatment of autoaggressive diseases, such as multiple sclerosis. The experimental allergic encephalomyelitis (EAE) respected animal model for MS was used for the study.
The aim of the study was the evaluation of the effect of pig spinal cord protein hydrolysate given orally upon
the ultrastructural changes in the blood-brain barrier image in EAE.
Changes of EAE are as follows: opened channels from basal membrane (tight junction) on the border
with astrocytes, fragments of organelles of the cells, oedema of astrocytes, presence of vesicles with
fluid, presence of macrophages with phagolysosomes. After pre-treatment with spinal cord hydrolysate
up to 6 weeks all the above changes were normalised.
These findings are promising as a possible tool in the clinical treatment of sclerosis multiplex.
key words: blood-brain barrier, oral tolerance, multiple sclerosis, experimental allergic encephalomyelitis,
autoaggressive diseases
INTRODUCTION
The aim of this study was to determine the effect
that spinal cord protein hydrolysate given orally has on
blood-brain barrier (BBB) changes in experimental allergic encephalomyelitis (EAE).
EAE is the accepted animal model for multiple sclerosis, an autoimmune disease of the central nervous
system.
Address for correspondence: Barbara Kwiatkowska-Patzer, PhD
Department of Neurochemistry,
Medical Research Centre, Polish Academy of Sciences,
ul. Pawińskiego 5, 02–106 Warsaw, Poland
tel. (48 22) 608 65 45; fax (48 22) 668 55 32
e-mail: [email protected]
The main target antigens for the immunological system in MS are thought to be: myelin basic protein —
MBP, myelin-associated glycoprotein — MAG, myelin oligodendroglial glycoproteins — MOG. Oral application of
these antigens and their components in small doses
suppress the immunological response.
The mechanism involved in oral tolerance is clonal
deletion, clonal anergy and active suppression.
The oral tolerance method is based on the thesis that
proteins are only partially hydrolysed and digested and partially different peptide fragments are crossing the jejunumblood barrier. Hypothetically there exists a system diminishing peptide antigenecity. It is known that lymphocytes T
CD8 are involved in the mechanism of suppression.
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Folia Neuropathol., 2003, Vol. 41, No. 1
Previous experiments were done with MBP and were
mildly successful [1].
Our suggestion is to evoke tolerance on the wide
spectrum of antigens in spinal cord proteins hydrolysate.
Suppression of immunological response diminishes clinical and histopathological signs of EAE [2, 4].
We undertook an ultrastructural study to establish
if the BBB changes seen in EAE disappear after treatment of EAE with spinal cord proteins hydrolysate given orally.
MATERIAL AND METHODS
Animals
Six- to eight-week-old female Lewis rats were housed
in animal care facilities and maintained on standard
laboratory chow and water ad libitum.
Ten female Lewis rats (200–250 g) were used in
the experiments. Two animals served as control group
and eight were divided into two groups — one with EAE
and the second with EAE pre-treated with oral spinal
cord protein hydrolysate.
Induction of CR-EAE
The Lewis rats were immunised in each hind foot
pad with 100 ml of solution containing guinea pig spinal
cord 50% homogenate in phosphate buffered saline and
complete Freund’s adjuvant containing 11 mg per 1 ml
pasteurised Mycobacterium phlei. The animals were
evaluated every other day. Scores were graded as follows: 0 — no symptoms, 1 — limp tail, 2 — hind leg weakness, 3 — hind leg weakness and incontinence, 4 —
paraplegia and weight loss, 5 — death.
Induction of oral tolerance
The animals were fed every day for 40 days with
5 mg of pig spinal cords proteins hydrolysate (preparation by Pharm. Lab. Dr A. W. Lipkowski) using syringe
with 20-gauge ball-point needle [2]. The control group
received 0.5 ml of water.
Ultramicroscopic study
On the 40th day post inoculation the rats were anaesthetised and perfused with 2% paraformaldehyde
and 2.5% glutaraldehyde in 0.1 M cacodylate buffer.
Specimens were taken from the hippocampal area
and fixed in the same solution for 20 hours and postfixed in a mixture of 1% Os04 [3] and 0.8% K4FeCN6.
Ultrathin sections were analysed in Jem 1200 JEM
Fx electron microscope.
Ultrastructural observations were concerned with
endothelial cells, perivessels space, astrocytes and
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macrophages and connective tissue elements. 100 electrograms were analysed from the hippocampal area.
Results were presented semiquantitatively.
RESULTS AND DISCUSSION
Lewis rats with experimental allergic encephalomyelitis show in the endothelial cells a number of pinocytic
vesicles and opened tight junctions (Fig. 1).
In the perivascular space there was present protein-rich fluid pressing perivascular cells. Basal membrane was wider with elements of proliferation. These
data indicate that in EAE synthesis of fibronectin
could be increased and as a main component of basal
membrane can be transferred into intracellular
space.
There was also evidence of oedema of astrocytes
(Fig. 2).
Myelin-like bodies were seen in endothelial cells
beside degenerative changes in them (Fig. 3).
Under basal membrane were found some collagen
fibrils (Fig. 4) as a sign of the repair process. We interpret these findings as evidence of the repair process at
the border between capillaries and the surrounding
brain cells. We discuss the hypothesis that the non-fibroblastic cells present in rat brain are able to synthesise collagen, which leads to a process comparable with
fibrosis of parenchymal organs [3, 5].
We also observed the brain macrophages with rich
lysosomal structures.
Increased perivascular brain phagocytes were observed in total anoxia in a way similar to our recent study
of experimental allergic encephalomyelitis (Fig. 5) [6].
In the examined animals after hydrolysate treatment
none of the changes described above was observed
(Table 1).
CONCLUSION
In experimental allergic encephalomyelitis morphological changes were observed in the blood-brain barrier
Table 1. Ultrastructural changes in blood-brain barrier
EAE
EAE
after
hydrolysate
+++
–
Pinocytic vesicles and damaged organellae +++
±
Perivessel fluid. Astrocytes oedema
+++
–
Increased activity of macrophages
+++
+
Collagen fibrils in perivessel space
++
–
Endothelium: tight junctions opened
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Barbara Kwiatkowska-Patzer et al., Influence of spinal cord protein hydrolysate...
Figure 1. Pinocytic vesicles (p) and opened tight junctions (arrow head) in the endothelial cells. In the perivascular space there is
a protein rich fluid (F) pressing perivascular cells. Bar 1 mm.
Figure 2. Opened tight junction. Evidence of oedema of astrocytes (OE). Bar 500 nm.
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Folia Neuropathol., 2003, Vol. 41, No. 1
Figure 3. Degenerative changes in endothelial cell. Opened tight junction. Myelin-like body (M) inside the endothelial cytoplasm.
Bar 200 nm.
Figure 4. Number of pinocytic vesicles and opened tight junctions in endothelial cells. Some collagen fibrils (C) under basal
membrane (as a reparative process). To the BBB is attached the macrophage with rich lysosomal structures (L). Bar 500 nm.
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Barbara Kwiatkowska-Patzer et al., Influence of spinal cord protein hydrolysate...
Figure 5. After hydrolysate treatment (6 weeks). In perivascular zone we observed normal neurones (N). Bar 1 mm.
Figure 6. After hydrolysate treatment (6 weeks). Microglial cells (G) in vessels region. Endothelial cells are normal. Bar
1 mm.
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Folia Neuropathol., 2003, Vol. 41, No. 1
as follows: opened tight junctions, pinocytic vesicles, oedema of astrocytes, presence of macrophages with phagolysosomes, fluid rich in protein in the perivascular space
pressing on pericyte.
After 40 days of oral treatment with pig spinal cord
hydrolysate all the changes described above had been
diminished or normalised.
These findings give the clinical implication that pig
spinal cord hydrolysate given orally could be successfully used as a tool in the treatment of multiple sclerosis in humans.
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