CEBPD is a protein that is made during inflammation in the spinal cord. The process of inflammation is required to heal injuries and may have beneficial as well as harmful effects. After the spinal cord is injured, the process of inflammation may damage relatively healthy nerves that are minimally injured in the initial incident. Also, inflammatory processes may retard new nerve growth and lead to scarring (mediated by astrocytes), blocking the way for new connections. This research project is aimed at determining if removing CEBPD; from inflammatory cells in an animal model of spinal cord injury will lead to the harnessing of the benefits of inflammation without the nerve damaging effects.
Since commencing the project in June 2006, equipment for the preparation of tissue for microscopic analysis has been purchased and is operational. A postdoctoral scientist and research assistant have been employed and have been trained. They have been performing animal experimentation and analysis for some time.
After the commencement of funding, I raised an additional $120,000 for specialised microscope equipment. These funds were raised by application to the Ramaciotti Foundation in addition to UNSW Faculty and St Vincent’s Hospital funding mechanisms. Due to a number of technical issues this equipment has only become fully operational in the last 4 months. However, substantial progress has been made in an effort to fulfill the aims of the project.
Aim-1 Define the locus, time course and cellular targets of neurodegeneration in the murine SC.
A colony of CEBPD; mice has been rederived after being imported from the USA. The animal model of immune induced spinal cord injury has been established in Australia. The altered disease severity in CEBPD; deficient mice has been confirmed and tissue examined by fluorojade. Now that we have overcome the technical difficulties with the new equipment, analysis is now underway and will be completed in the near future. Additionally, we have developed a specific polyclonal antibody, reacting against CEBPD; and validated its specificity. With this antibody we have defined the expression of CEBPD; throughout the course of EAE. Using double label immunohistochemistry, we have shown that CEBPD; is expressed in multiple cell types at different times throughout the course of immune induced spinal cord injury.
Early in disease CEBPD; is expressed in inflammatory cells called dendritic cells that modulate inflammation and are an absolute requirement to allow other immune cells to access to the CNS and induce disease. Later in disease, CEBPD; expression is found in the same cell type, with the addition of astrocytes. Astrocytes expressing CEBPD; are located at areas of prominent neurodegeneration and inflammatory cell infiltration. It is important to note that astrocytes are the main cell type causing scaring in the central nervous system. Consequently, our findings may have implications for repair of injury in addition to immune induced neurodegeneration
Aim-2 Determine the effect of modulating CEBPD; expression on neurodegeneration. We have established that neurodegeneration is decreased in animals that have decreased CEBPD; expression. Additional analysis has revealed that there are differences in inflammatory cell infiltration in areas where neurodegeneration are found in CEBPD; deficient animals.
While conducting this program of research, it has become clear that CEBPD; is a major transcription factor in the TNF-a inflammatory axis and mediates the transmission of signals from the IL-17 receptor. T-cells that produce IL-17 are the cells responsible for inducing neuronal damage in immune induced spinal cord injury. Our findings that reduced C/EBPδ leads to reduced spinal cord injury directly links altered neurodegeneration to changes in the immune response in immune induced spinal cord injury.
We have successfully isolated immune cells, astrocytes and microglia. These isolated cells will be used for microarray experiments outlined in the proposal in the coming year. Data from these experiments will assist in determining important factors associated with decreased neurodegeneration in CEBPD; deficient animals. Additionally, information gained from these experiments will help further determine the role of CEBPD; in spinal cord injury.
Aim-3 Examine the role of CEBPD; and the cellular milieu in neurodegeneration in SC injury.
Bone marrow transplant experiments have been conducted on two separate occasions giving similar results. The maximal effect of CEBPD; modulation is obtained when circulating inflammatory cells have no CEBPD;, with resident brain cells continuing to produce CEBPD;. In this case there is less inflammation and less spinal cord neurodegeneration than animals that are completely deficient in CEBPD;. Consequently, these experiments indicate that there is a protective effect of maintained CEBPD; expression in spinal cord glial cells. This phenomenon makes CEBPD; an especially attractive therapeutic target as the inflammatory cells that cause spinal cord injury can be targeted outside the CNS in the circulation.
Now that we have defined the cells that express CEBPD;, we are developing invitro assays to ascertain the direct effects of alteration of CEBPD; expression in individual cell types (immune, glia and neuronal). These experiments will complement microarray experiments and help ascertain important molecules mediating the effects of CEBPD; upon immune induced spinal cord injury.
2006-2007: CEBPD is a protein that is made during inflammation in the spinal cord. The process of inflammation is required to heal injuries and may have beneficial as well as harmful effects. After the spinal cord is injured, the process of inflammation damages relatively healthy nerves that are minimally injured in the initial incident. Also, it may retard new nerve growth and lead to scarring, blocking the way for new connections. This research project is aimed at determining if removing CEBPD from inflammatory cells in an animal model of spinal cord injury will lead to the harnessing of the benefits of inflammation without the nerve damaging effects.
Since the commencement of funding in June 2006, I have raised an additional $120,000 for specialised microscope equipment. These funds were raised by application to the Ramaciotti Foundation in addition to UNSW Faculty and St Vincent¡¦s Hospital funding mechanisms. This equipment will be operational for the analysis of spinal cord tissue from experiments early-2007.
The equipment for the preparation of tissue for microscopic analysis has been purchased and is operational. A postdoctoral scientist and research assistant have been employed and are currently undergoing intensive training. They will be responsible for some aspects of the animal studies and preparation of spinal cord tissue for analysis.
I completed bone marrow transplant experiments in the USA earlier this month, and processed the tissue and brought it back to my laboratory in Australia. Further experiments are currently being performed on this tissue. The current results indicate that bone marrow transplant with cells that lack CEBPD leads to appreciably less spinal cord nerve damage caused by inflammation. This raises the possibility that specialised bone marrow transplantation therapy represents an early option for the treatment of spinal cord injury. These results will have to be confirmed in repeat animal experiments.
Dr David Brown
MBBS (hons) PhD FRACP FRCPA
– NRMA Insurance / SpinalCure Australia Senior Fellow
– NH&MRC Postdoctoral Fellow
– Conjoint Senior Lecturer
Inflammation Research Laboratory
Centre for Immunology
St Vincent’s Hospital
University of New South Wales