This study is the first to examine the effects of oxaliplatin on the myenteric neurons in a mouse model of chemotherapy. Numbers and nNOS-IR of myenteric neurons were compared under two conditions: (1) ex vivo tissue culture with oxaliplatin for 3 and 24 h; and (2) repeated in vivo oxaliplatin administration into mice for up to 3 weeks. This allowed some neurotoxic effects of oxaliplatin on enteric neurons over short and extended periods of oxaliplatin administration to be evaluated and correlated with chronic oxaliplatin effects on colonic motility, in vitro.
Our major findings are consistent with an earlier study of the effects of cisplatin on rat enteric neurons (Vera et al., 2011
). Like cisplatin in rats, oxaliplatin in mouse causes a proportional increase of NOS-IR neurons and a significant reduction in the total number of myenteric neurons. The decrease in numbers of neurons correlates with a reduction in CMMC frequency, something seen previously in mouse models of Hirschsprung disease (Roberts et al., 2008
Dorsal root ganglion (DRG) neurons undergo apoptosis following exposure to oxaliplatin (Ta et al., 2006
; Scuteri et al., 2009
) and oxaliplatin accumulation in DRG neurons have been found (Holmes et al., 1998
; Cavaletti et al., 2001
; Ta et al., 2006
). This has been proposed as the underlying cause of oxaliplatin-induced sensory neuropathy (Gill and Windebank, 1998
). Platinum-based chemotherapeutic agents are designed to target and destroy proliferating cancer cells; but these agents also damage neurons, which are differentiated and non-proliferative. It has been suggested that platinum compounds may cause neurons to unsuccessfully re-enter the cell cycle (Gill and Windebank, 1998
), as cell cycle protein expression is altered following exposure to cisplatin (Gill and Windebank, 1998
; McDonald et al., 2005
). Platinum-induced apoptosis has also been associated with the involvement of the mitogen-activated protein kinases (MAPK) family (Scuteri et al., 2009
), which are responsible for regulating a number of cellular activities such as differentiation, proliferation, and cell death (Lewis et al., 1998
). In particular, the extracellular signaling-regulated kinase (ERK 1/2) and p38, which have well-recognized roles in neuronal apoptosis, have displayed early activation in DRG neurons following exposure to both oxaliplatin and cisplatin (Takeda and Ichijo, 2002
; Scuteri et al., 2009
Although the mechanisms of platinum-induced neuronal death have yet to be studied in enteric neurons, it has been speculated that the death of sensory DRG neurons involves afferent neurons innervating the gastrointestinal tract (Vera et al., 2011
). However, the extent of neuronal loss seen in our study suggests that oxaliplatin might also induce apoptosis of myenteric neurons which needs to be further studied. The death of enteric neurons is associated with impairment of colonic motility observed in our study and in a previous study with cisplatin (Vera et al., 2011
), which may explain some gastrointestinal symptoms experienced by patients.
Further oxaliplatin-induced changes to myenteric neurons in this study included the significant increase in the proportion of NOS-IR neurons both ex vivo
and in vivo
. NOS, and in particular nNOS, seems to play a significant role in oxaliplatin-induced neurotoxicity. This is noteworthy, as nNOS is typically expressed by interneurons and inhibitory motor neurons that supply the circular and longitudinal muscles of the gastrointestinal tract (Bornstein et al., 2004
). A recent study has shown that the repeated administration of oxaliplatin in the rat causes an upregulation of nNOS in the spinal cord (Mihara et al., 2011
). This finding has been associated with the incidence of mechanical allodynia, the sensation of pain caused by a stimulus that would not normally produce pain, e.g., light touch or cold. The administration of both selective and non-selective NOS inhibitors prevented the pain behavior initially seen in rats following oxaliplatin administration.
The production of NO by nNOS depends on levels of cytoplasmic Ca2+
. When cytoplasmic Ca2+
is elevated, nNOS is activated, possibly leading to excessive NO production, which causes cell damage (Yagihashi et al., 2000
), and may account for the oxaliplatin-induced increase in NOS-IR neuron size seen in this study. This needs to be further investigated. Elevated cytoplasmic Ca2+
is also known to cause cytotoxic damage to neurons and can trigger apoptosis (Rivera et al., 2011
Conversely, the increased proportion of NOS-IR neurons seen with oxaliplatin administration in our study could partly be due to the loss of other subpopulations of myenteric neurons. Therefore, these results could be due to a combination of both increases in cytoplasmic Ca2+
and free-radical NO, and the death of other neuronal subgroups within the ENS. It is also possible that NOS-IR neurons are capable of better survival following oxaliplatin administration than other subgroups of myenteric neurons, as nNOS neurons have been shown to remain quite resistant to toxicities in Alzheimer's disease, Huntington's disease, and vascular stroke (Gonzalez-Zulueta et al., 1998
). Recent study in nNOS knockout mice has shown that NO produced by nNOS has protective effects on enteric neurons following intestinal ischemia/reperfusion injury (Rivera et al., 2012
). Further studies need to be carried out investigating the mechanisms by which oxaliplatin-induced changes occur in the enteric neurons and determining which subpopulations are being affected. The effect of oxaliplatin on enteric neuron function also needs to be examined, including changes in the electrophysiological properties of neurons, neurotransmission, and ion channel functions.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.