Title of the project: “Cellular and molecular mechanisms of

regeneration in zebrafish spinal cord”

(BT/PR5489/AAQ/03/245/2004).

Project Investigator and Address: Dr. Sukla Ghosh, Department of

Biophysics, Molecular Biology and Genetics, University of Calcutta,
92, APC Road Kolkata-700009.

Introduction: Adult Zebrafish shows spontaneous recovery after CNS injury

unlike mammals. Any injury to mammalian spinal cord leads to a complex series
of cellular and molecular responses. There is overwhelming inflammatory
response, which trigger several other secondary tissue damage, neuronal and
glial loss, progressive cavitation and scarring, which would contribute to
regeneration failure and significant functional loss in mammals.
Although zebrafish has emerged as a powerful animal model to study
regeneration but little attention has been paid to study spinal cord regeneration in
this species. All of the previous studies on other teleost spinal cord have been
focused on either giving complete transection or after tail amputation and none
of these have examined the response to crush, hemisection or contusion injuries,
which are known to be of significant focus of research efforts in mammalian
models. In our present study we have used two different injury models crush and
transection so that data can be compared to results of the contemporary studies
on mammalian modes of SCI (spinal cord injury). The goal of study is to
understand the cellular and molecular events underlying the regenerative
processes in order to gain insights into probable novel therapies for inducing
regeneration in higher vertebrates including human.

Results:
A. Characterization of injury: We have standardized two different injury models
and studied time course analysis of the regenerative process by histological
techniques, like H&E, trichrome, Luxol Fast Blue and Cresyl Violet. Fig.1 shows
the extent of axonal and neuronal regeneration at different time points after SCI.
We have demonstrated substantial axonal regeneration after 1 month. Earlier we
have characterized the cellular events which involve infiltration/inflammation,
ependymal sealing, accumulation of astrocytes in injury site and subsequently
regeneration of both grey and white matter without forming any astrocytic scar.
B. Cell proliferation: Successful regeneration of spinal cord requires both
axonal regeneration and replacement of lost neural tissue. We have
demonstrated that neurogenesis occurs in adult Zebrafish uninjured cord (fig.3A),
which is thought to be a unique phenomenon, using BrdU/PCNA
immunohistochemistry. In order to replace the lost tissue after injury a high level
of proliferation/ neurogenesis is required (Zupanc et al 2005). We have studied
the time course analysis of cell proliferation after SCI. Fig.3 shows the rate of
BrdU incorporation in injured and uninjured spinal cord. There is an induction of
proliferation at day 3 (Fig.3B), followed by a very high level of proliferation at day
7 and subsequently the rate of proliferation begins to decrease at day10 after
SCI (data not shown). The rate of proliferation at day 15 post injury levels with
normal uninjured cord (data not shown). We have confirmed our results with
other proliferation markers. Thus spinal cord injury induces proliferative response
both in white matter and grey matter but predominantly in ependymal cells
(Fig.3C), many of which are radial glia and are thought to be the neural
precursors.
C. Axonal regeneration and role of inhibitory molecules: To understand the
extent of axonal regeneration we have studied time course analysis of
regenerating cord at different time points. Transected cord showed no axonal
sparing at 7 day post injury where we can see regenerative response resulting in
sealing of the ependyma but still the two cut ends of the spinal cord are
physically apart confirming no axonal sparing (Fig.4A). Axonal regeneration is
evident at the site of injury as shown by Luxol Fast Blue staining (Fig.1B) in both
transection and crush injury. There is substantial axonal regeneration in 1 month
regenerated spinal cord (Fig.1B) which resulted in functional recovery as well
(fig.2) and was recorded by reappearance of swimming behavior after initial
paralysis (observed just after spinal cord injury).
Failure of axonal regeneration in mammalian cord is due to accumulation of toxic
myelin breakdown products like myelin associated glycoprotein (MAG),
Oligodendrocyte-myelin glycoprotein (OMgp ) which are thought to be inhibitory
in their action. The Nogo-66 receptor (NgR), which can also bind to MAG and
OMgp, has been proposed to play an important role in the mediation of axonal
growth inhibition (Fournier, GrandPre, and Strittmatter 2001; Liu et al. 2002).
Previous hypothesis was that absence of Nogo-66 and its receptor in
regeneration competent species may allow axonal regeneration to proceed. But
recently Nogo and its receptor have been cloned in zebrafish (Klinger et al 2004).
We have demonstrated a strong level of expression of NgR in normal brain
(Fig.4B&4F) and are presently studying the expression in uninjured and injured
spinal cord.

Thus functions of NgR proteins in fish are likely to differ from their mammalian
function as transducer of growth inhibition. So it raises an intriguing question
whether fish cord do express NgR after injury and what its function might be in
these regeneration competent species after SCI as compared to mammals. We
are presently studying expression of MAG and NgR (Nogo-66 receptor) after SCI
in zebrafish in order to gain further insight into regeneration permissive
environment and role of Nogo-66 receptor NgR and MAG.

References

1. Fournier, A. E., GrandPre,T.´, and Strittmatter,S.M. (2001) Identification of a

receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 409:341–
346.
2. Liu, B. P., Fournier,A., GrandPre,T.,´, and Strittmatter,S.M. (2002). Myelin-
associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science
297:1190–1193.
3. Zupanc, G.K., Hinsch, K., Gage, F.H. (2005) Proliferation,migration,neuronal
differentiation,and long-term survival of new cells in the adult Zebrafish brain. J
Comp Neurol 488:290-319.
Summary:

Zebrafish is an established model in regeneration research and can

spontaneously recover from CNS injury, unlike mammals. This phenomenon has
not been studied adequately. We have standardized two different injury models
e.g. crush (commonly used and reported in mammalian spinal cord regeneration
research) and transection in zebrafish spinal cord to elucidate the cellular and
molecular mechanism involved during regeneration. Cellular proliferation data
indicate a low level of neurogenesis even in uninjured adult cord and induction
of a very high proliferative response after crush injury. This neurogenesis
phenomenon enables zebrafish to recover anatomically and functionally from
spinal cord injury which we have shown. No previous studies have been done to
characterize the cellular events after crush injury and to study proliferative
response in cord after crush injury in zebrafish.

We have also demonstrated axonal regrowth after spinal cord injury as evident
by swimming behavioural recovery analysis and role of axonal inhibitory
molecules like Nogo-66 receptor (NgR). We showed expression of NgR in
zebrafish CNS reflecting its role in regeneration competent species which may be
different from what we see in mammals.

Technologies transferred: Not applicable

Patent: Not applicable
Publications: Subhraprakash Hui, Amitava Chatterjee and Sukla Ghosh 2008.
Regeneration of spinal cord in zebrafish: cell proliferation versus cell death. -
Manuscript in preparation.
Relevant photographs: Please refer to attached figures.