Characterization of ATP Nanoliposome Treatment for Regeneration of Injured Spinal Cord
Traumatic spinal cord injury (SCI), due to automobile accidents, falls, or sport injuries, is a significant global public health concern. Following intracellular delivery of adenosine triphosphate (ATP) nanoliposomes or gamma-thio ATP nanoliposomes and immunohistochemical staining with antibodies to CD 68 and CD 163, the effects of treatment of injured rat spinal cord sections can be examined. ATP is the energy currency of the cell. A method to measure ATP and its metabolites (ADP and AMP) in cells treated with ATP-encapsulated nanoliposomes is outlined. The detection limit using HPLC and UV absorption for ATP is 0.1 micromole. The method is applicable for characterizing the lipid encapsulation, leakage and degradation of ATP-laden nanoliposomes as a potential treatment for accelerated wound healing has been extensively studied in a rabbit model of wound healing. This method is also applicable to studying the energy state of the cells in culture and how this is influenced by treatment with ATP nanoliposomes with varying lipid composition. The provision of a supplemental intracellular ATP supply is evaluated to determine the structural integrity of the spinal cord (as judged by the spinal cord histology of the white and gray matter). Immunohistochemical analysis is performed to assess whether there is greater proliferation of the regenerative M2 (CD68+ and CD163+) macrophage population compared to the M1 (CD68+ and CD163−) population. Examination of spinal cord sections could determine whether candidates like the ATP nanoliposomes either alone or in combination with an anticomplement agent could have a potential benefit in regeneration and accelerating functional recovery following injury.
KeywordsSpinal cord injury Regeneration of injured spinal cord ATP nanoliposomes Histology Immunohistochemical analysis Intracellular ATP estimation
The authors would like to thank Dr. Bernard Moss of the NIAID, NIH for providing us with the initial stock of RK-13 cells and Arkdadiusz Slawomir Slusarczyk from the group of Dr. Martha Bickford, Department of Anatomical Sciences, University of Louisville School of Medicine for expert assistance with electronmicroscopy of the nanoliposome formulations and Dr. Harshul Pandit for his assistance in suggesting initial HPLC methods. The embedding of frozen spinal cords, sectioning and hematoxylin and eosin staining was performed in the special procedures lab of the Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine by Ms. Melissa Peak. The spinal cord injury with the weight drop method and the microinjection was carried out by Ms. Christine Yarberry from the Kentucky Spinal Cord Injury Research Center (KSCIRC) at the University of Louisville School of Medicine, Louisville, KY. The staining of the spinal cord sections with Nissyl/Cresyl violet for the gray matter and Eriochrome cyanine for the white matter was carried out by Ms. Alice Shum-Siu from the laboratory of Dr. David Magnusson at the KSCIRC. This work was supported in part by grants DK74566, AR52984, HL114235, GM106639, DK104625, DK105692, and OD021317 from the National Institutes of Health and in part from the Kentucky Cabinet for Economic Development, Office of Entrepreneurship, under the Grant Agreement KSTC-184-512-12-138 and KSTC-184-512-14-174 with the Kentucky Science and Technology Corporation.
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