We developed a high throughput, objective, operant assay for orofacial function and validated its ability to detect acute and chronic functional allodynia. The dolognawmeter quantified a behavioral index of nociception in three separate mouse models of orofacial pain (TMJ inflammation, masticatory myositis and head and neck cancer). To confirm that the behavioral dependent measure (gnaw time) reflects nociception, we restored gnawing function by blocking nociception with the same analgesics used clinically for patients with comparable pathology. Our assay with the dolognawmeter provides a metric for orofacial function and pain that can now be used to investigate molecular mechanisms in the trigeminal system and test analgesics for treating pain in the head and neck.
Our assay exploits a rodent’s instinctual response to gnaw through an obstruction in a narrow tube as first described by Ayada et al. (Ayada et al., 2002
). These authors measured gnawing rate by quantifying the mass
of plastic gnawed from a hard plastic confinement strip perpetually replaced on the end of a narrow tube over a fixed period of time
; the task is infinite. Our device employs an automated mechanism that records the amount of time
required for a mouse to gnaw through an obstruction and actually gain escape. This entails a discrete gnawing task
because the mouse always gnaws though the dowel in a similar pattern after training and the device retracts both ends of the dowel from the tube the instant that it is severed. While confined in the dolognawmeter, the animal is unable to turn around but there is no direct restraint as seen in some assays (Amir and Amit, 1978
; D’Amour and Smith, 1941
; Randall and Selitto, 1957
Gnawing is a routine, physiologic orofacial function. It is coordinated by the trigeminal somatosensory and motor systems and activates the TMJ, craniomandibular muscles, jaws, incisors, lips, tongue, buccal mucosa, palate and gingiva in a fashion that is similar to the chewing associated with mastication. Functional allodynia originating from pathology in any of these structures is potentially quantifiable with the dolognawmeter. Reflexive orofacial nociceptive assays are less applicable to musculoskeletal nociception generated by function since they often induce an acute, transient painful stimulus in the skin to produce allodynia (Morgan and Gebhart, 2008
). In addition most operant orofacial nociceptive assays do not measure orofacial dysfunction resulting from orofacial pathology but rather from noxious cutaneous stimulation (Neubert et al., 2008
). In our device, routine voluntary orofacial function involving most of the masticatory complex produces nociception resulting from a clinically relevant pathology such as masseter myositis or oral cancer. An ethical advantage of our device and assay is that the animal controls the stimulus exposure. Thus, this method appropriately follows IASP and NIH guidelines (Zimmermann, 1983
The dolognawmeter measures functional mechanical allodynia in the rodent orofacial complex. Mechanistically, the source of nociception is likely a combination of chemical and mechanical for all three models studied. The anatomic structures involved in the three models can be inferred from the site of injury used to create those models. For the TMJ inflammation model the source of nociception is the joint where CFA was injected. The TMJ is a closed space surrounded by dense fibrous connective tissue and extravasation from the joint into surrounding tissues is unlikely. The mechanical allodynia likely occurs during loading of the joint when the mouse is gnawing. Injection of CFA or carrageenan into a joint has been widely used as a model of inflammatory hyperalgesia in articular tissue and produces functional changes consistent with arthritis in patients (Hutchins et al., 2000
; Kehl et al., 2000
; Tonussi and Ferreira, 1992
, , 1999
). Intramuscular CFA injection produces a valid model for the investigation of muscle hyperalgesia (Harriott et al., 2006
). For the masticatory myositis model CFA is injected directly into the masseter muscle. The source of nociception is likely nociceptors within the muscle or the periosteum overlying the mandible. To confirm nociception in the TMJ arthritis and masseter myositis models we completely antagonized the effect of CFA injection on gnawing function by pre-administering a systemic NSAID. We confirmed the infiltrate histologically (i.e. neutrophil infiltrate (Harper et al., 2001
)) in both the TMJ and masseter inflammatory models. For the oral cancer model the carcinoma proliferates within the submental space. The submental space is bounded by muscle, periosteum and bone. The source of nociception within the cancer model could be any or all of these structures. We validated the dolognawmeter with these three distinct models of orofacial pathology to demonstrate that the instrument detects orofacial nociception originating from different tissues within the orofacial region. The device can quantify and the investigator can compare dysfunction and thus nociception resulting from different pathologies.
The dolognawmeter possesses a unique combination of experimental and technical advantages. Experimentally, it measures both acute and chronic nociception. We measured nociception hours and days following CFA administration and weeks following SCC inoculation. Assays that quantify stereotyped behaviors such as rubbing and flinching of the head have also been used as an index of acute orofacial nociception; however, these assays are generally subjective, low throughput and less effective for measuring chronic nociception (Roveroni et al., 2001
). Various meal size and interval assays have been proposed to quantify both acute and chronic nociception (Harper et al., 2001
; Harper et al., 2000
; Kerins et al., 2005
; Thut et al., 2007
). Feeding assays are potentially confounded by variables that affect appetite including analgesics, systemic disease, time of day, duration of the study, and reward associated with consumption. For example, animals with cancer can weaken, cease eating, and develop cachexia in the absence of nociception. Feeding studies cannot resolve behavioral changes due to orofacial nociception versus nociception originating elsewhere in the body since mice with non-trigeminal nociception demonstrate reduced appetite and feeding rate (Kerins et al., 2005
). Appetite is less likely to affect the outcome variable in the dolognawmeter because animals are confined for a relatively brief period in the tube without food. Moreover, the gnawing behavior that we measure does not involve consumption even though it engages most of the orofacial complex used for mastication. The mouse does not consume debris from the dowel since the animal occludes the oral cavity with the tongue, cheeks and lips behind the incisors while gnawing.
High throughput is a technical advantage of the dolognawmeter; the device is fully automated and multiple devices can be run concurrently. This study was undertaken using 30 dolognawmeters. Measurement of the outcome variable is objective and not prone to investigator bias. To accommodate instinctual proclivities of the mouse, a nocturnal prey animal, the experiment takes place in darkness in the absence of the investigator. Initial delay due to habituation, animal loading inconsistencies, or drug side effects is less likely to affect the outcome measure since only the gnaw times for the second dowels are compared. An animal can delay gnawing on the first dowel until pharmacologic sedation has worn off and/or analgesia has taken effect without influencing the gnaw time for the second dowel. Thus, the device accommodates pharmacokinetic differences between animals and pre-empts intractable difficulties with pharmacologic sedation, titration and analgesic onset.
A pharmacologic motor effect is less likely to corrupt the outcome variable since the first dowel acts as a prerequisite minimum motor verification task. The dolognawmeter is not prone to false positives when evaluating the efficacy of sedating analgesics since sedation and analgesia produce opposite test outcomes. In most reflexive assays, sedation and analgesia produce a similar test result. For example, in the paw withdrawal assay, sedation and analgesia both increase withdrawal threshold. Without a motor verification task, operant trials such as the mouse facial-thermal-operant assay are likely to produce a false negative when evaluating sedating analgesics since the sedative effect alone can attenuate the operant behavior (Neubert et al., 2008
We validated measurement of nociception in both cancer and inflammatory models. Using the head and neck cancer pain model we demonstrated oral dysfunction following cancer inoculation. Reduced function secondary to carcinoma-induced nociception was also observed in our previous work demonstrating progressive increase in withdrawal threshold and a decrease in weight bearing in the carcinoma-injected hindpaw mouse model (Schmidt et al., 2007
). In both our hindpaw model and the current head and neck cancer model we partially restored function with high dose morphine, the first line treatment for cancer pain (Schmidt et al., 2007
The dolognawmeter has potential limitations. Non-nociceptive factors can influence gnawing activity. Systemic illness can weaken an animal and increase gnaw time since gnawing requires effort. We demonstrated only partial recovery of gnawing function in animals with head and neck cancer. This could be due to a number of variables including subtotal analgesia or systemic illness. Complications concerning systemic disease might be ameliorated with a lower durometer dowel that poses an easier gnawing task. However, gnaw times are comparable only when using dowels of the same durometer.
Potential behavioral confounders include analgesic side effects such as gnawing stereotypy. Opiates and other dopaminergic drugs induce gnawing stereotypy under specific conditions requiring upright position of the animal or an environment that permits gnawing assisted climbing (Tirelli and Witkin, 1995
). The dolognawmeter precludes these conditions and thus prohibits gnawing stereotypy induced by these drugs (Livezey et al., 1995
; Tirelli and Witkin, 1995
). Morphine administered to naïve mice did not potentiate gnawing activity in the dolognawmeter.
The dolognawmeter quantified an index of nociception to demonstrate functional allodynia in three orofacial conditions. It may also be useful to study painful disorders that are physiologically unique to the orofacial region including toothache, trigeminal neuralgia, and headache. Because the device activates and measures goal-directed behavior, complex diseases such as depression and anxiety could also be studied. We anticipate that future studies will test the validity and identify the shortcomings of the dolognawmeter for these and other diseases.