Birds That Sing and Die: Beta Amyloid Precursor Protein as a Marker for Avian Traumatic Brain Injury
Abstract
Accurate information regarding the morbidity and mortality of zoo animals is important in their captive management and may affect the success of efforts to reproduce critically endangered species. Traumatic injury is one of the leading causes of death in avian zoological specimens and head trauma is a major subset of these cases. In veterinary patients, the ability to diagnose traumatic brain injury is often hampered by incomplete history and a lack of gross lesions or histologic evidence visible by routine staining and light microscopy. The diagnosis of traumatic brain injury is often tentative and made by exclusion of other causes. Accurate, rapid, and economic methods to document injury to the brain would provide an important tool in avian diagnostics.
In the last 10 years there has been a surge of research regarding the pathophysiology, diagnosis, and treatment of traumatic brain injury in humans.1,8 Recent advances have indicated the importance of axonal injury (AI) as a cause of morbidity and mortality. AI in humans and mammalian models can be detected with high specificity and sensitivity by immunohistochemical demonstration of Pamyloid precursor protein (P-APP).2,3,5,7,9 A neuronal glycoprotein, P-APP, is carried by rapid anterograde transport within the axon and accumulates in areas where that transport is impaired (i.e., sites of axonal injury). These immunohistochemical methods have been used successfully in forensics to document many kinds of mechanical injury, such as “shaken baby syndrome,” with as short a posttraumatic survival period as 30 minutes.2,9 To date, no published efforts have been made to apply these methods to nonmammalian species, though the presence of P-amyloid has been confirmed in an avian model4 and a single avian patient6.
The hypothesis of this study was that P-APP accumulates within damaged axons in the brains of birds which have died from traumatic brain injury, and that this P-APP can be detected by immunohistochemical methods already developed for mammalian species.
Case material was gathered from archived necropsy cases of the Zoological Society of San Diego. Sixteen adult birds were examined of which three had histologically apparent axonal lesions, and the others had either confirmed gross, histologic, clinical, or historic evidence compatible with head trauma. Two cases also had spinal cord trauma. Species included representatives from six orders (Columbiformes, Psittaciformes, Apodiformes, Passeriformes, Ciconiformes, and Gruiformes).
Three birds euthanatized for unrelated reasons were used as negative controls. Human brain tissue from a patient with Alzheimer’s disease was used as a positive control. An additional 14 cases from the archives of the ZSSD in which the birds died suddenly and no diagnosis was found were also examined. Using standard immunohistochemical avidin-biotin complex techniques, two antibodies against P-APP were utilized at varying concentrations: a) P-APP (clone 22, Cl 1; Boehringer AG, Mannheim, Germany),2,3,5,7,9 and b) P-APP C-terminus (CT 695; Zymed Laboratories, 561 Eccles Ave. So., San Francisco, CA 94080 USA)9.
The results of this study confirmed that P-APP is produced in bird neural tissue, that it does accumulate within damaged axons, and that immunohistochemical techniques developed for its detection in mammalian subjects will work in avian species. Subjectively, the use of P-APP is helpful in detecting injured axons that might otherwise be overlooked in standard H&E sections. In addition, the results of at least one case indicate that immunohistochemical staining with P-APP may detect axonal injury earlier than standard histopathologic techniques. AI, however, was not detected in the majority of confirmed head trauma patients in this study and AI was not found in any “sudden death” cases in a population of birds which died suddenly of unknown causes. These negative results most likely reflect some of the limitations of this study, since relatively few serial sections were examined in each case and standardization of the anatomic location, posttrauma to death interval, or character of the original traumatic event was not possible. The subjective increase in neuronal staining with P-APP may also be important, but these results could not be confirmed objectively without carefully matched controls. While further work utilizing cases with a welldefined trauma to death interval is needed to fully develop this diagnostic tool, P-APP immunohistochemical staining can be a potentially important new method to diagnose and study acute brain injury in avian species.
Acknowledgments
The authors thank Anthony Adame and Yvonne Cates, HT (ASCP) for their technical expertise, Dr. Brian Summers for initial consultation, Drs. Ilse Stalis and Rebecca Papendick for assistance in identifying suitable cases, and the UCSD Experimental Neuropath Laboratory and the Zoological Society of San Diego for their support.
Literature Cited
1. Blumbergs, P.C., N.R. Jones, and J.B. North. 1989. Diffuse axonal injury in head trauma. J. Neurol. Neurosurg. Psychiatry. 52:838–841.
2. Finnie, J.W. and P.C. Blumbergs. 2002. Animal models: Traumatic brain injury. Vet. Pathol. 39:679–689.
3. Gleckman, A.M., M.D. Bell, R.J. Evans, and T.W. Smith. 1999. Diffuse axonal injury in infants with nonaccidental craniocerebral trauma. Arch. Pathol. Lab. Med. 123:146–151.
4. Louzada, P.R., A.C.P. Lima, F.G. deMello, and S.T. Ferreira. 2001. Dual role of glutamatergic neurotransmission on amyloid 1–42 aggregation and neurotoxicity in embryonic avian retina. Neurosci. Lett. 301:59–63.
5. Medana, I.M., N.P. Day, T.T. Hien, N.T.H. Mai, D. Bethell, N.H. Phu, J. Farrar, M.M. Esiri, NJ. White, and G.D. Turner. 2002. Axonal injury in cerebral malaria. Am. J. Pathol. 160:655–666.
6. Nakayama, H., K.-I. Katayama, A. Ikawa, K. Miyawaki, J. Shinozuka, K. Uetsuka, S.-I. Nakamura, N. Kimura, Y. Yoshikawa, and K. Doi. 1999. Cerebral amyloid angiopathy in an aged great spotted woodpecker (Picoides major). Neurobiol. Aging. 20:53–56.
7. Oehmichen, M., C. Meibner, V. Schmidt, I. Pedal, H.G. Konig, and K.-S. Satemus. 1998. Axonal injury—a diagnostic tool in forensic neuropathology? A review. Forensic Sci. Int. 95: 67–83.
8. Reilly, P.L. 2001. Brain injury: the pathophysiology of the first hours. “Talk and Die revisited.” J. Clin. Neurosci. 8:398–403.
9. Stone, J.R., R.H. Singleton, and J.T. Povlishock. 2000. Antibodies to the C-terminus of the [3-amyloid precursor protein (APP): a site-specific marker for the detection of traumatic axonal injury. Brain Res. 871:288–302.