Transcript Analysis of the effects of Streptomyces hyaluronidase on formation of the neural tube Gary C.

Analysis of the effects of
Streptomyces hyaluronidase on
formation of the neural tube
Gary C. Schoenwolf and Marilyn Fisher
From the Department of Anatomy, University of Utah
Journal of Embryology and Experimental Morphology
Bethany Rovnack
December 4, 2003
Neurolation is the complete
development of the neural tube
The process that initiates the formation
of the nervous system
Neurolation occurs in 3 steps:
1. Formation of the Neural Plate
2. Bending of the Neural Folds
With Formation of the Neural Groove and Neural Folds
Elevation of the Neural Folds
3. Convergence of the Neural Folds
Fusion of the Neural Folds
Background:
•Experimentation on the development of
the neural tube began about
50 years ago
•Different hypotheses have been
presented and debated
•Intrinsic: concentrated to neural plate
•Extrinsic: forces outside the
neural plate
• Studied the role of Hyaluronic Acid
is contained within the extracellular
matrix
• It is found under neural folds of both
avian and mammal embryos
• Hyaluronic acid makes up about
84% of the glycosominoglycans
(GAGs) made by the tissues when
the neural tube is first starting to
develop
Chick embryo of
thirty-three
hours’
incubation
Background cont…
•Hyaluronic acid exists naturally in all
living organisms and is a universal
component in the extracellular matrix.
• It provides a cushion effect
•Provides protection.
• Same chemical structure bacteria as
well as in human beings.
• It is produced by tissues in early
embryos, such as the neural tube.
Background Continued…
•Hyaluronic Acid plays an important role in tissue hydration,
lubrication and cellular function.
•It is able to hold more water than any other natural substance.
•Due to its hydrating properties, it increases smoothness, and
softening and decreased wrinkles where it is found.
Reasonable to Conclude that…
Because Hyaluronic Acid is involved in hydration, with increased
volume in the neural folds within the extracellular matrix, the force
produced will would aid in the production of the neural tube
(neurolation).
Would Neurolation occur normally in embryos that lacked
Hyaluronic acid?
Hypothesis:
If there was a decrease in the amount
of Hyaluronic acid in the embryo, then
neurolation would not occur normally,
leading to an increase in the number of
neural tube defects (NTD).
To Test the Hypothesis:

Chick Embryos in ovo treated with
Streptomyces hyaluronidase
Materials & Methods:
Embryos (stages 8-9) studied for formation of spinal cord
• Special precautions were taken to study effects only
caused by Streptomyces hyaluronidase only
• Other side effects could hinder the study - they were taken
into account
FYI: Hyaluronic Acid is part of the family of enzymes,
which inhibits the growth of the glycosaminoglycan
chain(GAGs) through the plasma membrane into the
extracellular matrix.
•All neural tube defects found in the embryo’s developing
spinal cords were those treated with Streptomyces
hyaluronidase due to the failure of increase in neural folds
1. White Leghorn Chicken eggs were incubated @ 38˚C
2. A small window was cut through the part of the shell that
covered the embryo using standard techniques
3. The following was injected into the sub-blastodermic space of the 4
different test embryos:
(a) 20ul SH (200 TRU/ml in 0.9% saline) – Batch 1 & 2
(b) 20ul SH in a solution of 3 parts: 0.9% saline to 1 part
albumen, trypsin
(c) 20ul Hyaluronic Acid digested with
40 TRU, SH & boiled 5 min
to destroy the enzyme, 0.9% saline
(d) 3 parts 0.9% saline to 1 part albumen
4. All embryos were illuminated with fiber optics
5. The “windows” were sealed with scotch tape
6. Returned to incubator for an additional 24h.
Assay for Protease Activity
Reason: An assay was done to determine the level of
protease contamination contained within the
batches of Streptomyces hyaluronidase used
in the treatment of the embryos.
Fibrinogen/agar-coated Plate I was prepared using the following procedure:
1.) 1g agar in 75 ml – 0.05m Tris-Buffer
2.) 0.014m Calcium Lactate was boiled for 15 min, or until agar
dissolved
3.) Cooled to 60ºC
Fibrinogen/agar-coated Plate II was prepared using the following procedure:
1.) 80mg Fibrinogen in 25ml of 0.025m Tris-Buffer
2.) Centrifuged
3.) Slowly added to the agar
Both mixtures were incubated for 1h in 80 ºC
Poured into a 5ml/dish
- cooled to room temperature
studied right away
storied in the refrigerator
- Small wells were made in the newly formed agar/fibrinogen
gels using a glass using a glass pipette.
-Filled with 10ul of enzyme solution
- Incubated @ 37ºC for 24-36h
-Prepared for Processing of Microscopy
Summary of the various concentrations of enzymes tested
for proteolytic Activity
Agents Tested
Streptomyces Hyaluronidase
Pronase or Trypsin
Pronase or Trypsin +
Albumen
Concentrations
200 TRU/ml
100ug/ml
10ug/ml
1ug/ml
0.1ug/ml
0.01ug/ml
0.001ug/ml
100ug/ml
10ug/ml
1ug/ml
0.1ug/ml
Detectable Lysis
No
Yes
Yes
Yes
Barely (@36h)
No
No
Yes
No
No
No
Discussing the Effects:
• Most embryos treated with Streptomyces
hyaluronidase experienced NTD
•The frequency found in this particular
study was related to the batch of enzymes used
•NTD formed in 60% of the embryos treated with E1
• 94% of the embryos treated with E2
Experienced neural tube defects
•However, no major difference could be detected in
the two batches of enzymes (E)
•They both appeared to be equally effective in their
activity within the matrix
Histology
Gross Features of NTD:
Treatment of embryos with Streptomyces hylaronidase
resulted in embryos experiencing “non-closure” type NTDs:
(1) Failure of convergence
(2) Unsuccessful fusion along the spinal cord
(3) The amount of enzymatic activity
Distinguishing Characteristics
• Reduction in the size of the extracellular space
• Marked dilation of the embryonic blood vessels
• Presence of intracellular inclusions
Solutions injected
subblastodermically
# if embryos with # of embryos with
closed neural
neural tube
tubes
defects
% of embryos
with neural tube
defects
Hyaluronidase
(1)
24
36
60
Hyaluronidase
(2)
3
51
94
Hyaluronidase
(2) + saline
0
16
100
Trypsin
(0.1ug/ml)
14
2
13
Hyaluronic Acid
digested with
hyaluronidase (2)
and boiled for 5m
24
3
11
Saline/Albumen
Mixture
64
10
14
Fig. 30: EMF-induced abnormalities in
chicken embryos.
(Farrell et al, 1997)
Pictures (a)-(d) show normal,
unexposed embryos following a 48 hour
incubation. The spinal cord is the "tail"
of the embryo, and the neural
l tube inside this appears completely
normal; so does the developing
brain. Picture (h) is a transverse section
at the level of the hindbrain
for embryo (a) as indicated by line (h)
in picture (a). This brain is
normal.
Pictures (e)-(g) and (i) show neural tube
and brain defects induced by
electromagnetic fields. In (e)-(g)
arrows indicate neural folds of open
neural tubes in the spinal cord. This is
an abnormality known in humans
as spina bifida. The hindbrain shown in
(i) of embryo (g) is
abnormal - totally flattened - compared to the
normal brain shown in (h).
1. Elevation of the Neural Folds
2. Convergence of the Neural Folds
3. Fusion of the Neural Folds across the
Dorsal midline
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
CONSEQUENCES:
•The depletion of SH has no effect on elevation
•However, convergence is blocked completely when
Streptomyces hylaronidase is absence
•As well as the inhibition of fusion of the neural folds
Conclusion:
The results presented support
the idea that neurolation is a complex process involving several
different components. The important role of the extracellular
matrix is key in the data shown above. The experiments displayed
here show that the effects of neurolation due to the injections
were not solely due to the protease contaminate nor the
Hyaluronic Acid. This is supported by the data of those embryos
that were treated with the Hyaluronic Acid and did not experience
any deficiencies. Only 14% of the control group experienced
NTD. The hypothesis stated in the literature supports the theory
that multiple factors are most likely the main factor of
neurolation.
Analysis of the effects of Streptomyces hyaluronidase on the formation of the neural tube
By Gary C. Shoenwolf and Marilyn Fisher
From the Department of Anatomy, University of Utah
Journal of Embryology and Experimental Morphology
73, 1-15. 1983