A mouse model of pharyngeal dysphagia in amyotrophic lateral sclerosis.

We recently established that the SOD1-G93A transgenic mouse is a suitable model for oral-stage dysphagia in amyotrophic lateral sclerosis (ALS). The purpose of the present study was to determine whether it could serve as a model for pharyngeal-stage dysphagia as well. Electrophysiological and histological experiments were conducted on end-stage SOD1-G93A transgenic mice (n = 9) and age-matched wild-type (WT) littermates (n = 12). Transgenic mice required a twofold higher stimulus frequency (40 Hz) applied to the superior laryngeal nerve (SLN) to evoke swallowing compared with WT controls (20 Hz); transgenic females required a significantly higher (P < 0.05) stimulus frequency applied to the SLN to evoke swallowing compared with transgenic males. Thus, both sexes demonstrated electrophysiological evidence of pharyngeal dysphagia but symptoms were more severe for females. Histological evidence of neurodegeneration (vacuoles) was identified throughout representative motor (nucleus ambiguus) and sensory (nucleus tractus solitarius) components of the pharyngeal stage of swallowing, suggesting that pharyngeal dysphagia in ALS may be attributed to both motor and sensory pathologies. Moreover, the results of this investigation suggest that sensory stimulation approaches may facilitate swallowing function in ALS.

An animal model of oral dysphagia in amyotrophic lateral sclerosis.

Relatively little is known about the underlying neuropathology of dysphagia in amyotrophic lateral sclerosis (ALS); thus, effective treatments remain elusive. Tremendous progress toward understanding and treating dysphagia in ALS may be possible through the use of an animal model of dysphagia in ALS research; however, no such animal model currently exists. The most logical candidate to consider is the SOD1-G93A transgenic mouse, the most widely investigated animal model of ALS. To investigate whether this animal model develops dysphagia, oral behaviors (lick and mastication rates) of SOD1-G93A transgenic mice (n = 30) were evaluated at three time points based on hind limb motor function: asymptomatic (60 days), disease onset (approximately 110 days), and disease end-stage (approximately 140 days). Age-matched nontransgenic littermates (n = 30) served as controls. At each time point, lick and mastication rates were significantly lower (p < 0.05) for transgenic mice compared with controls. Histologic analysis of the brainstem showed marked neurodegeneration (vacuolation) of the trigeminal and hypoglossal nuclei, two key motor components involved in mastication and licking behaviors. These results demonstrate a clinicopathologic correlation of oral dysfunction in SOD1-G93A transgenic mice, thereby establishing the SOD1-G93A transgenic mouse as a bona fide animal model of oral dysphagia in ALS.

Role of soluble guanylate cyclase in the trigeminal subnucleus caudalis in capsaicin-induced muscle hypersensitivity.

Nitric oxide (NO) produces its effects by activating soluble guanylate cyclase (sGC). In the present study, we investigated the potential role of sGC in the subnucleus caudalis (Vc) in mediating masseter hypersensitivity under acute inflammatory condition in male Sprague-Dawley rats. First, our Western blot analysis revealed that sGC protein is reliably detected in the Vc. Subsequent immunohistochemical studies demonstrated that neuronal cell bodies in the superficial laminae of the Vc positively stained for sGC. Astrocytes in deeper lamina of the Vc also showed sGC immunoreactivity. We then tested whether intrathecal administration of sGC inhibitors, methylene blue (MB), and ODQ, in the Vc, attenuates masseter hypersensitivity induced by intramuscular injection of capsaicin. Intrathecal MB or ODQ significantly blocked the capsaicin-induced reduction of mechanical threshold to noxious stimulation of the masseter. These data indicate that the NO-sGC pathway in the Vc is involved in mediating orofacial muscle hypersensitivity under acute inflammatory conditions.

Hypertonic saline-induced muscle nociception and c-fos activation are partially mediated by peripheral NMDA receptors.

In this study, the animal model of hypertonic saline (HS) infusion protocol was developed and utilized to test the hypothesis that HS causes peripheral release of glutamate, and that blockade of peripheral NMDA receptors significantly reduces HS-induced nocifensive behavior and central neuronal activation. Nocifensive behavior and c-fos immunoreactivity, as a marker of central neuronal activation, were assessed from the animals that received intramuscular HS infusion with and without the NMDA receptor antagonist, MK-801. HS infusion (20 microl/min for 10 min) in the rat masseter produced prolonged nocifensive hindpaw shaking responses that peaked in the first minute and gradually diminished over the infusion period. The HS induced nocifensive behavior was dose-dependently attenuated by MK-801 pretreatments (0.3 mg/kg and 0.1 mg/kg), but not by vehicle pretreatment (isotonic saline; ISO), in the masseter muscle. HS infusion produced a significant number of Fos positive neurons in the ispsilateral subnucleus caudalis (Vc). Subsequent immunohistochemical studies showed that peripheral MK-801 pretreatment effectively reduced the HS induced neuronal activation in the Vc. These results provide compelling evidence that HS-induced muscle nociception is mediated, in part, by peripheral release of glutamate, and that blockade of peripheral glutamate receptors may provide effective means of preventing central neuronal activation.

The influence of pain on masseter spindle afferent discharge.

Muscle spindles provide proprioceptive feedback supporting normal patterns of motor activity and kinesthetic sensibility. During mastication, jaw muscle spindles play an important role in monitoring and regulating the chewing cycle and the bite forces generated during mastication. Both acute and chronic orofacial pain disorders are associated with changes in proprioceptive feedback and motor function. Experimental jaw muscle pain also alters the normal response of masseter spindle afferents to ramp and hold jaw movements. It has been proposed that altered motor function and proprioceptive input results from group III muscle afferent modulation of the fusimotor system which alters spindle afferent sensitivity in limb muscles. The response to nociceptive stimuli may enhance or reduce the response of spindle afferents to proprioceptive stimuli. Several experimental observations suggesting the possibility that a similar mechanism also functions in jaw muscles are presented in this report. First, evidence is provided to show that nociceptive stimulation of the masseter muscle primarily influences the amplitude sensitivity of spindle afferents with relatively little effect on the dynamic sensitivity. Second, reversible inactivation of the caudal trigeminal nuclei attenuates the nociceptive modulation of spindle afferents. Finally, functionally identified gamma-motoneurons in the trigeminal motor nucleus are modulated by intramuscular injection with algesic substances. Taken together, these results suggest that pain-induced modulation of spindle afferent responses are mediated by small diameter muscle afferents and that this modulation is dependent, in part, on the relay of muscle nociceptive information from trigeminal subnucleus caudalis onto trigeminal gamma-motoneurons. The implication of these results will be considered in light of current theories on the relationship between jaw muscle pain and oral motor function.

Assessing mechanical sensitivity of masseter muscle in lightly anesthetized rats: a model for craniofacial muscle hyperalgesia.

In this report, we present a simple and reliable way of assessing mechanical sensitivity of masseter muscle as a model for craniofacial muscle hyperalgesia. Mechanical thresholds that evoke nocifensive hindpaw responses following noxious masseter stimulation were assessed. Masseteric injections of widely used sensitizing agents significantly increased mechanical sensitivity of the muscle in a time dependent manner without affecting other muscles and overlying skin. This lightly anesthetized rodent paradigm allows us to provide calibrated and reliable mechanical stimulus, which is not possible in behaving animals. The technique can be applied to study mechanistic bases for craniofacial muscle tenderness.

Innocuous jaw movements increase c-fos expression in trigeminal sensory nuclei produced by masseter muscle inflammation.

Muscle tenderness and pain during movements are prominent symptoms associated with persistent jaw muscle pain. However, there is virtually no information on how trigeminal neurons respond to jaw movements (JM) or muscle palpation in the presence of muscle tissue injury or myositis. In this study, we investigated the effects of innocuous JM in the presence of acute masseteric inflammation on postsynaptic responses in the trigeminal brainstem nuclei by examining the expression of c-fos. In one group of rats, unilateral injections of an inflammatory substance, mustard oil (MO: 20%, 25 microl) were made into a masseter muscle. In another group, controlled and systematic JM were provided following MO injection. Three additional groups of rats were used to control for anesthetic, JM, and injection procedure. MO injected in the masseter muscle induced a high level of Fos protein expression in four principal trigeminal regions: the subnucleus caudalis (Vc), the ventral and dorsal regions of the Vc/Vi (subnucleus interpolaris) transition zone, and the paratrigeminal nucleus (PTN). Movements following MO injection consistently produced a significantly greater level of Fos expression in all these areas, especially in the Vc/Vi transition region and caudal Vc on the ipsilateral side. Importantly, movements also induced a significantly greater level of Fos expression in the caudal Vc on the contralateral side. The present results provide the first documentation that innocuous JM in the presence of muscle inflammation significantly increase the MO-induced c-fos expression in the trigeminal brainstem nuclei, which may explain the greater pain experienced during movement of inflamed or injured muscles.