Effectiveness of Rolipram Administration to Enhance Endogenous Neural Precursor Cell

Proliferation for Spinal Cord Injury Repair

By:

Chelsea Cosner

Lucia Herrmann

HHMI High School Summer Scholars Program

PI: Dr. Michael Gaines

Thursday, July 29, 2010

Mentor:

Dr. Damien Pearse

Miami Project to Cure Paralysis

Lois Pope Life Center

Abstract

The objective of this project was to experiment with Rolipram and see if its

administration to rats that suffered a spinal cord injury would decrease levels of

phosphodiesterases (PDEs) and subsequently increase neural progenitor cell numbers in stem

cell niches, namely the rostral migratory stream (RMS), subventricular zone (SVZ), and

enendymal layer of the spinal cord. The benefit of lower levels of PDEs would be an increase in

cyclic AMP (cAMP). After a spinal cord injury, higher levels of cAMP would promote axonal re-

growth and create a better environment for progenitor cells to proliferate and differentiate into

other needed cells within the brain. The rats treated with Rolipram (1.25 mg/Kg/d) dissolved in

15% ethanol-saline did indeed show a decrease in PDE4B and an increase in proliferation of

progenitor and glial progenitor cells. Altered neuronal and astrocytic differentiation between the

test and control subjects was not evident by qualitative review.

Introduction

Spinal cord injury (SCI) is a devastating condition affecting approximately 180,000–

230,000 individuals in the United States, with 11,000 new cases annually. The majority of

reported human injuries, nearly 40%, occur at the cervical level, producing compression or

contusion of the spinal cord. Two major pathological phases occur in SCI: a primary mechanical

injury, producing immediate cell death and tissue damage, and a secondary injury phase,

initiated by the first and characterized by progressive destruction on the spinal cord tissues from

the lesion epicenter, the site of the injury, that occurs over subsequent hours to weeks, resulting

in tissue cavation (Figure 1). Until recently, it was generally thought that there was little or no

useful regeneration or repair after spinal cord lesions in adult mammals. The discovery,

however, of neural precursor cell populations within distinct regions of the central nervous

system that are able to generate new neural cells, including neurons, may provide a therapy for
spinal cord injury repair. Potentially, this could improve the quality of life of injured patients. The

current work seeks to develop a pharmacological treatment that targets the enhancement of

neural progenitor proliferation after SCI to facilitate both neuroprotection and replacement so

that functional restitution can be achieved.

Figure 1. Hematoxylin, eosin and luxol fast blue stained 10 µm transverse sections from the injury
epicenter, and at 600 and 1500 µm rostral (above the injury site) and caudal (below the injury site) from a
representative spinal cord at 9 weeks after and severe cervical injury. Significant differences in the lateral
and longitudinal extension of tissue damage can be seen among injury severities. (Modified from Pearse
et al., 2005)

Previous studies have shown that Rolipram, a phosphodiesterase 4 (PDE4) inhibitor,

can enhance the proliferation of endogenous- naturally present- progenitor cells (Nakagawa et

al., 2002). The PDE gene family includes enzymes that substrate cyclic adenosine

monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels within the brain

and central nervous system (CNS). The presence of PDE4B and PDE4D are inhibitory to

cAMP, while PDE5A, is inhibitory to cGMP. As cyclic nucleotide levels have been shown to

regulate proliferation and differentiation, PDE4 and PDE5 could play an essential role in these

processes within the neural progenitor of the central nervous system. As an important

secondary messenger, cAMP has also been shown to reduce cell death and atrophy and

increase axonal re-growth and repair after spinal cord injuries. Cyclic AMP leads to the release

of calcium, an important neurotransmitter and promotes gene expression whose products lead

to direct axonal regeneration (such as arginase I and interleukin-6) (Hannila et al - 2008). It also

counteracts the effects of myelin-inhibitors such as myelin-associated glycoprotein (MAG),

oligodendrocyte myelin glycoprotein (OMgp) and ephrin B3, which is one of the main problems

associated with spinal cord injuries.

 Neural progenitor cells are widespread throughout the central nervous system. In this

work, we focused on the progenitors residing in the subventricular zone (SVZ), as the most

research has been done on them, making them good candidates to easily asses Rolipram

administration effects on their proliferation. These progenitors of the brain travel along the

rostral migratory stream (RMS), with their final destination being the olfactory bulb (Figure 2).

Generally, neural progenitors can develop into astrocytes, oligodendrocytes, microglial cells, or

neurons. Depending on the type, or differentiation stage, of these progenitors, they can express

Nestin (all progenitors), GFAP (progenitors/astrocytes), NG2 (glial progenitors), or PSA-NCAM

(future neurons). The presence of PDEs can be gauged from these markers under fluorescent

microscopy as PDEs inhibit the cAMP that fosters an environment for progenitors and glial

progenitors as well as the subsequent markers they produce. Decreased markers mean less

progenitors and glial progenitors and their products, such as neurons, oligodendrocytes and

astrocytes. This can mean more PDEs are present. Contrastingly, a brain that shows many of

these markers has new neurons and support cells being produced and may have inhibited

levels of PDEs and higher levels of camp.

Figure 2. Progenitor cells in different areas of SVZ give rise to specific types of neurons. CC, corpus
callosum; Ctx, cortex; SVZ, subventricular zone; RMS, rostral migratory stream; OB, olfactory bulb; TH,
tyrosine-hydroxylase; CR, calretinin; CB, calbindin. (Modified from Whitman et al, 2009).

The objective of our project was to validate the effectiveness of Rolipram administration
by analyzing the effects on PDE4 expression within the injured spinal cord and by detecting

increases in proliferation and differentiation of progenitor cells within the SVZ, RMS and

ependymal layer of the spinal cord. Therefore, the project includes the staining with antibodies

to brain samples to observe various indicators of proliferation and differentiation for these cells.

This is a part of larger projects in the lab and will serve to consolidate a proper model for further

studies.

Materials and Methods

Cervical contusion injury and drug administration

Adult female Fischer rats (180– 200 g) were housed according to NIH and The Guide for

the Care and Use of Animals. Under ketamine (45 mg/kg i.p.) and xylazine (5 mg/kg, i.p.)

anesthesia a laminectomy-the removal of a vertebrae- was performed at cervical vertebra C5.

The cervical contusion injury was created using the Electromagnetic SCI Device developed at

Ohio State University. The exposed C5 spinal cord was moderately injured by displacement of

the spinal cord by 0.95 mm. After injury, the muscles were sutured in layers and the skin was

closed with sutures. The rats were allowed to recover in a warmed cage with water and food

easily accessible. Gentamicin (5 mg/kg, intramuscular) and the analgesic Buprenex (0.01 mg/kg

of 0.3 mg/mL, subcutaneous) were administered daily. The rats were maintained for 3 days. The

Institutional Animal Care and Use Committee of the University of Miami approved all animal

procedures.

Rolipram (1.25 mg/Kg/d) dissolved in 15% ethanol-saline was administered immediately

after the injury and every 8 hours, until the sacrifice (n=4). The control rats received 15%

ethanol-saline.

BrdU was administered at a dose of 50 mg/Kg/d 24 h before the injury and daily until the

sacrifice- this would allow for glia to be expressed under fluorescence microscopy.
Western blot

The western blots were performed by our supervisor to quantify the amount of PDE4A,

PDE4B, and PDE4D proteins in the spinal cord samples. Western blots show protein levels

within an environment. Bands indicate the presence of a protein and the further down a gel

band is, the smaller the amount of protein.

Tissue retrieval

At 3 days after injury rats were anesthetized (70 mg/kg ketamine, 10 mg/kg xylazine),

the C5 cervical spinal cord (5-mm-long piece) was dissected and immediately frozen in liquid

nitrogen. Rats were transcardially perfused to solidify and preserve their tissue with phosphate-

buffered, 4% paraformaldehyde (0.1 M, pH 7.4) and the brains were removed, post-fixed with

4% of paraformaldehyde and after stored in phosphate buffered-saline.

Histology

The brains were embedded in 30% sucrose and frozen for cryostat sectioning. The

forebrain was cut into 20 µ m thick sections. Fifteen series of 7 slides with 4 sections per slide

were attained.

The tissue was first stained for 5 minutes with Sudan Black to prevent autofluorescence

then washed 3 times with 1x TBS. Each tissue was placed in a cassette and then into

sequenzas and washed again with 1x TBS. The tissue was then blocked for non-specific

immunoglobulin binding in a 5% Goat Serum-TBS-0.3%Tx and left for an hour. The primary

antibodies, Ki67, to detect proliferating cells; SMI22, to detect astrocytes; PSA-NCAM, to detect

neuroblasts; Nestin, to detect progenitor cells, NeuN; to detect neurons; NG2, to detect glial

progenitors, as part of the binding antibodies were applied in 2.5% Goat Serum TBS-0.3%TX-

100 and stored at 4 ºC overnight. Then, the tissue was washed 5 times with TBS-Tween20 and

the secondary antibody labeled with Alexa fluorocroms (488 for green staining, or 594, for red
staining), were applied to the slides in TBS-0.3%TritonX-100, binding to the antibodies.

Additional washes with TBS-Tween20 were applied the next day followed by two 1x TBS

washes and one 1x TB wash. The slides were then coated with Vectashield and cover slipped.

Slides are to be stored at 4 ºC.

For BrdU labeling the sections were placed in 1M HCl for ten minutes on ice. Then, the

sections were changed to a 2M HCl solution for ten minutes at room temperature, and finally

placed in another 2M solution at 37ºC for twenty minutes. The sections were washed with 0.1M

borate buffer. After the borate buffer, the sections were washed with 1x TBS.

Fluorescence microscopy and picture acquisition

Images of the ventricles, SVZ and RMS were acquired with a fluorescent microscope

using the program Stereo Investigator at magnifications of 200x and 400x. Filter combinations of

DAPI (blue), TX RED (red), and EN GFP (green) were used. Pictures acquired were of the outer

ventricle and hippocampus. The outer ventricle showed the RMS, progenitors and their

products that were stained to show up in florescent.

Results

PDE4B expression was down regulated after Rolipram administration in SCI.

It is already published that after chronic treatment (fourteen days) with Rolipram, PDE4A

protein levels do not change, while PDE4B decreases and PDE4D increases (Dlaboga et al

-2006). In order to analyze if Rolipram administration was effective in our model, after three

days of administration, the protein levels of these different isoforms of PDE4 in the spinal cord

injured samples, treated with Rolipram or Ethanol, were quantified by western blot techniques.

The results were then graphed to compare the differing protein levels (Figure 3).
Figure 3. Diagram bars showing the levels of PDE4A, PDE4B and PDE4D after Ethanol or Rolipram
administration, related to actin levels.

The graphs obtained show that PDE4A and PDE4D levels were not changed after Rolipram

administration. However, the levels of the 78 KDa protein of the PDE4B was significantly

decreased for the animal treated with Rolipram.

Increased proliferation of progenitor cells after Rolipram administration in SCI

The antibodies Ki67 and BrdU were used to show proliferating cells, after tissue retrieval

and while the animals were still alive respectively. After tissue retrieval the antibody against

BrdU marked the proliferative and differentiated cells after the injury. Therefore, to see if

Rolipram was producing an increase of proliferation, we used the antibody against Ki67. Under

the microscope, there is an apparent increase in proliferation with an increased number of cells

expressing Ki67 in SVZ in the Rolipram treated animals, with respect to the ethanol treated

animals (Figure 4). To see if this proliferation was generating an increased number of
progenitors, antibodies against Nestin and NG2 were used. Staining showed no change in

Nestin marker levels, and therefore little indication of changed progenitor levels. It seems that

there is an increase of NG2- a marker for glial progenitors, although further quantization should

be done.

Figure 4. Diagram comparing the results of the Rolipram versus ethanol-saline treated rats. The Ki67 and
NG2 cells seem to be more numerous in the Rolipram animal and the levels of Nestin show no significant
changes when looking at the samples on the microscope.

In order to know if the increased number of cells were producing an increased number of

any specific cell type, immunohistochemistry against PSA-NCAM (cells that will be neurons) and

GFAP (astrocytes) was performed (Figure 5). We did not find qualitative differences on PSA-

NCAM, or GFAP, indicating that neuron and astrocyte levels did not change. Also BrdU-NeuN

and BrdU-GFAP staining was performed (Figure 5). Although an increase in BrdU positive cells

in Rolipram-treated animals versus ethanol-treated animals was found, the staining used was

not sufficient to indicate changes in the differentiation towards a specific cell type between

ethanol and Rolipram treatments.

 Figure 5. Diagram showing
increased levels of BrdU-
Neun, and BrdU-NeuN in
the Rolipram treated animal.
There was no major
qualitative difference
between the two animals
with the staining of PSA-
NCAM and GFAP when
taking all samples into
consideration.

Discussion

The results of western blot show that Rolipram administration after SCI was partially

effective. In previous cases where Rolipram was administered fourteen days (Dlaboga et al

-2006), as opposed to three, PDE 4B levels were decreased while PDE 4A levels remained

approximately the same. Our results with the western blot technique concurred with this

experiment. Other proteins affected by Rolipram should be analyzed to confirm the

effectiveness within the spinal cord. We qualitatively found a possible increase of proliferation

and generation of glial progenitors under microscopic investigation. However, we cannot say

qualitatively if the already generated cells are producing a specific cell type or if they are

differentiating to all cell types. The data should be quantified for more specific results that
indicate if specific cells are being produced or if the differentiation is ubiquitous. Staining for

oligodendrocytes was not performed and should be taken into consideration for future

experiments.

In conclusion, Rolipram administration in the spinal cord injury model used seems to be

effective in increasing neural progenitors proliferation, although a quantization of the staining

would further support that conclusion.

Acknowledgements:

We would like to thank the Miami Project and specifically Dr. Damien Pearse, Dr. Clara

Penas, Samik Patel, and Armstrong Ibe for their assistance and support of our research. We

could not have written this paper without them. We would also like to thank Gus Arriolla, Jesus,

Navid, Sebastien, Dimitri, Zachary, Abraham and Amored for making our experience at Pearse

Lab an unforgettable one. We would also like to thank Dr. Gaines and the HHMI Program for

High School Scholars for giving us this opportunity.

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