Isolation, characterization and pressure response of equine hoof keratinocytes
The horse's hoof is a mechanical structure that has the ability to respond to changes in the way it is loaded. One type of response is for the external shape of the hoof capsule to alter, thereby changing the mechanical behavior of the whole hoof in response to externally applied loads. The hoof also has the ability to respond pathologically leading to failure due to disease and injury. Laminitis, or inflammation of the hoof wall, is one of the top two diseases identified by the American Association of Equine Practitioners affecting horses today. This dissertation investigated methodologies to isolate keratinocytes extracted from the coronary band of the hoof. These cells were then subjected to increased pressures, either statically or intermittently, using a novel stress reactor built specifically for this project. Equine hoof keratinocytes (EHK) were evaluated to determine relationships between apoptosis, proliferation and gene expression in response to increasing levels of compressive pressure ' in vitro'. A new optimal methodology was achieved to obtain and subculture keratinocytes from the hoof. Immunocytological analysis of EHK revealed a keratin expression pattern indicative of nail cells. EHK responded to pressure with altered levels of proliferation and apoptosis, indicating that these cells can detect and respond to pressure 'in vitro' as they do 'in vivo'. Following pressurization, cells were evaluated for alterations in regulatory gene expression. Intermittent pressure (IP) affected cells by decreasing levels of cyto-protective Akt gene expression and the stress-activated protein c-Jun N-terminal kinase (SAPK/JNK) while levels of the SAPK/p38 were elevated. Following administration of the p38 inhibitor SB203580, IP induced cell death was abolished in EHK with no effect on proliferative potential. In addition, inhibition of p38 induced an increase in SAPK/JNK expression, indicating a relationship between p38 and JNK in EHK subjected to IP. Elevations in p38, along with increased apoptosis, have been associated with laminitis in the past. This dissertation reveals that SAPK/p38 may have a regulatory role in EHK pressure response and manipulation of this pathway, along with an analysis of downstream signaling, may be the key to understanding hoof maladies and disease progression thereby advancing potential therapeutic benefits for horses.