Ocular surface-evoked Fos-like immunoreactivity is enhanced in trigeminal subnucleus caudalis by prior exposure to endotoxin.

Endotoxin-induced uveitis (EIU) is a common animal model for anterior uveitis in humans that causes long-term changes in trigeminal brain stem neurons. This study used c-fos immunohistochemistry to assess the effects of different routes of administration of endotoxin on activation of trigeminal brain stem neurons produced by ocular surface stimulation. A single dose of endotoxin (lipopolysaccharide (LPS)) given to male rats by systemic (i.p., 1 mg/kg) or intraocular (ivt, 20 microg) routes increased the number of Fos-positive neurons in rostral (trigeminal subnucleus interpolaris/subnucleus transition (Vi/Vc)) and caudal portions of trigeminal subnucleus caudalis (trigeminal subnucleus caudalis/upper cervical spinal cord transition (Vc/C(1-2))) by 20% mustard oil (MO) applied to the ocular surface 7 days, but not at 2 days, after LPS compared with naïve rats. I.c.v. (20 microg) LPS did not affect MO-evoked Fos. To determine if the pattern of enhanced Fos expression after systemic LPS also depended on the nature of the ocular surface stimulus, additional groups received ocular stimulation by 10% histamine or dry eye conditions. Seven days, but not 2 days, after i.p. LPS both histamine- and dry eye-evoked Fos was increased at the Vi/Vc transition, while smaller effects were seen at other regions. These results suggested that EIU modulation of trigeminal brain stem neuron activity was mediated mainly by peripheral actions of LPS. Enhancement of Fos at the Vi/Vc region after MO, histamine and dry eye conditions supports the hypothesis that this region integrates innocuous as well as noxious sensory information, while more caudal portions of Vc process mainly nociceptive signals from the eye.

Estradiol replacement modifies c-fos expression at the spinomedullary junction evoked by temporomandibular joint stimulation in ovariectomized female rats.

The influence of estradiol (E2) treatment on temporomandibular joint (TMJ) nociceptive processing in the caudal trigeminal sensory brain stem complex was assessed in ovariectomized female rats by quantitative Fos-immunoreactivity (Fos-LI). After 2 days of daily injections of high (HE2) or low (LE2) dose E2 rats were anesthetized and the small fiber excitant, mustard oil (MO, 0-20%), was injected into the TMJ and after 2 h brains were processed for Fos-LI. TMJ-evoked Fos-LI in laminae I-II at the trigeminal subnucleus caudalis/upper cervical cord (Vc/C1-2) junction and the dorsal paratrigeminal region (dPa5) was significantly greater in HE2 than LE2 rats, while Fos-LI produced at the ventral trigeminal interpolaris/caudalis transition region (Vi/Vc(vl)) was similar. E2 treatment also modified the influence of N-methyl-D-aspartate (NMDA) and AMPA receptor antagonists on TMJ-evoked Fos-LI. The NMDA antagonist, MK-801, dose-dependently reduced the Fos-LI response at the Vc/C1-2 junction in HE2 rats, while only high dose MK-801 was effective in LE2 rats. MK801 reduced equally the Fos-LI response at the Vi/Vc transition in both groups, while only minor effects were seen at the dPa5 region. The AMPA receptor antagonist, NBQX, reduced Fos-LI at the Vc/C(1-2) and Vi/Vc(vl) regions in HE2 rats, while only high dose NBQX was effective in LE2 rats. NBQX did not reduce Fos-LI at the dPa5 region in either group. These results suggest that estrogen status plays a significant role in TMJ nociceptive processing at the Vc/C1-2 junction mediated, in part, through ionotropic glutamate receptor-dependent mechanisms.

Oestrogen receptor-immunoreactive neurons in the trigeminal sensory system of male and cycling female rats.

Many common craniofacial pain conditions are more prevalent in women than men and may be related to the phase of the menstrual cycle. Long-term effects of oestrogen in the nervous system are produced by receptor-mediated [oestrogen receptor alpha (ERalpha) and beta (ERbeta) isoforms] mechanisms; however, it is not known if the distribution of ER-positive neurons in the trigeminal system is similar in males and females. Quantitative immunocytochemistry was used to compare the distribution of ERalpha-labelled neurons in the trigeminal brainstem complex (TBC) and ganglion of male and female rats at different stages of the oestrous cycle. A high density of ERalpha-labelled neurons was seen in the superficial laminae (I-III) throughout the trigeminal subnucleus caudalis (Vc) and the upper cervical dorsal horn. Counts of ERalpha-positive neurons in laminae I-III were similar for prooestrous and dioestrous females, while males had fewer cells. The deeper laminae (IV-V) of the Vc and the cervical dorsal horn had few ERalpha-positive neurons in all groups. At the region surrounding the central canal at caudal levels of the Vc, prooestrous females had more ERalpha-positive neurons than dioestrous females or males. Few labelled cells were seen rostral to the trigeminal subnucleus interpolaris/caudalis transition region (Vi/Vc) in any group. In the trigeminal ganglion, prooestrous and dioestrous females had a moderate (8-10%) number of nuclear-labelled small or medium-sized neurons, while males had fewer labelled cells (4.5%). Qualitatively, the pattern of staining for ERbeta was similar, although weaker, than for ERalpha in the trigeminal dorsal horn or ganglion. These results were consistent with the hypothesis that oestrogen acts through trigeminal ganglion cells and caudal portions of the Vc to modulate sensory and autonomic aspects of craniofacial pain in a sex-related manner.

Topical cannabinoid agonist, WIN55,212-2, reduces cornea-evoked trigeminal brainstem activity in the rat.

Cannabinoids act at receptors on peripheral and central neurons to modulate diverse physiological functions and produce analgesia. Corneal sensory nerves express the CB1 cannabinoid receptor and project to two spatially discrete regions of the lower brainstem, the trigeminal interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical cord (Vc/C1) junction region. The function of CB1 expression on corneal nerves is not known. To determine if cannabinoid receptors in the anterior eye affect the activity of trigeminal brainstem neurons at the Vi/Vc and Vc/C1 the CB1 agonist, WIN55,212-2 (WIN-2), was applied topically prior to chemical excitation of corneal afferent fibers. In the first series of experiments WIN-2 was applied topically prior to excitation of corneal nociceptors by mustard oil (MO). WIN-2 reduced significantly the number of Fos-like immunoreactive neuronal nuclei (Fos-LI) at the Vi/Vc transition (-46.7+/-8.2%, P<0.05), while smaller non-significant reductions occurred at the Vc/C1 junction region (-20.3+/-7.6%). The selective CB1 antagonist, SR141716A (1mg/kg, i.v.), prevented WIN-2-evoked reduction in Fos-LI after MO. Systemic administration of WIN-2 (1 or 10mg/kg, i.p.) or SR141716A (1mg/kg, i.v.) or topical corneal application of morphine sulfate did not affect Fos-LI produced by MO. In parallel experiments, topical WIN-2 reduced the magnitude of single unit activity recorded at the Vi/Vc transition (-80+/-7%, P<0.025), but not at the Vc/C1 junction region (-34+/-30%) evoked by CO(2) pulses applied to the cornea. Topical morphine did not alter CO(2)-evoked unit activity at either recording location. These results indicated that cannabinoid receptor agonists acted, at least in part, at CB1 receptors in the anterior eye to reduce corneal stimulation-evoked trigeminal brainstem neural activity. Corneal nociceptor-evoked activity at the Vi/Vc transition was reduced significantly by topical WIN-2, while activity at the Vc/C1 junction region displayed only minor decreases. These findings were consistent with the hypothesis that CB1 receptors affect the activity of corneal-responsive neurons that preferentially contribute to homeostasis of the anterior eye and/or reflexive aspects of nociception rather than the sensory-discriminative aspects of corneal nociception.

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat.

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.

Morphine and NMDA receptor antagonism reduce c-fos expression in spinal trigeminal nucleus produced by acute injury to the TMJ region.

Pain management in temporomandibular disorders (TMDs) often involves pharmacotherapy; however, the site of action for drugs that reduce TMD pain is not known. To determine possible central neural targets of analgesic drugs relevant in TMD pain, morphine or the N-methyl-D-aspartate receptor antagonist, MK-801, was given alone or in combination prior to TMJ injury. The number of neurons expressing the immediate early gene, c-fos, was quantified in the lower brainstem and upper cervical spinal cord as an index of neural activation. It was hypothesized that those neuronal groups most necessary for the sensory-discriminative aspects of acute TMJ injury should display the greatest reduction in c-fos expression after drug treatment. Barbiturate-anesthetized male rats were given morphine or MK-801 15 min prior to injection of mustard oil into the TMJ region. Morphine given centrally (i.c.v.) or peripherally (i.v.) caused a marked dose-related reduction in Fos-like immunoreactivity (Fos-LI) in laminae I-II at the middle portions of subnucleus caudalis (mid-Vc) and at the subnucleus caudalis/upper cervical spinal cord (Vc/C2) transition. Higher doses of morphine also reduced Fos-LI in the dorsal paratrigeminal region (dPa5) and at the subnucleus interpolaris/subnucleus caudalis (Vi/Vc-vl) transition. MK-801 given i.v. reduced Fos-LI only in laminae I-II at the Vc/C2 transition. Combined subthreshold doses of morphine and MK-801 reduced c-fos expression in the dPa5, mid-Vc, and the Vc/C2 transition region, below that predicted from the effects of either drug alone. These results suggest that neurons in laminae I-II of the mid-Vc and Vc/C2 transition and, to a lesser extent, in the dPa5 region play a critical role in mediating the sensory and/or reflex aspects of pain after acute injury to the TMJ region.

Selective blockade of substance P or neurokinin A receptors reduces the expression of c-fos in trigeminal subnucleus caudalis after corneal stimulation in the rat.

Stimulation of the cornea activates neurons in two distinct regions of the spinal trigeminal nucleus: at the transition between trigeminal subnucleus interpolaris and subnucleus caudalis and at the transition between trigeminal subnucleus caudalis and the upper cervical spinal cord as estimated by expression of the immediate early gene, c-fos. To determine if receptors for substance P or neurokinin A, neurokinin 1 and neurokinin 2 receptors, respectively, contribute to the production of Fos-positive neurons in these brainstem regions, receptor-selective antagonists were given intracerebroventricularly 15 min prior to stimulation of the cornea in anesthetized rats. The number of Fos-positive neurons produced in superficial laminae at the trigeminal subnucleus caudalis/cervical cord transition by application of the selective small fiber excitant, mustard oil, to the corneal surface was reduced by the neurokinin 1 receptor antagonist, CP99,994 (5-100 nmol, i.c.v.) and the neurokinin 2 receptor antagonist, MEN10,376 (0.01-1.0 nmol, i.c.v.). Combined pretreatment with CP99,994 and the competitive N-methyl-D-aspartate receptor antagonist, CPP, caused a greater reduction in c-fos expression at the subnucleus caudalis/cervical cord transition than after either drug alone suggesting interaction between receptors for glutamate and substance P. Tachykinin receptor antagonists did not reduce the number of Fos-positive neurons produced at the subnucleus interpolaris/subnucleus caudalis transition. The elevation in plasma concentration of adrenocorticotropin, but not the increases in arterial pressure or heart rate, evoked by corneal stimulation was prevented by pretreatment with CP99,994 or MEN10,376 at doses lower than those needed to reduce c-fos expression. The results indicate that receptors for substance P and neurokinin A contribute to the transmission of sensory input from corneal nociceptors to brainstem neurons in trigeminal subnucleus caudalis and to increased activity of the hypothalamo-pituitary axis that accompanies acute stimulation of the cornea.

N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor antagonism reduces Fos-like immunoreactivity in central trigeminal neurons after corneal stimulation in the rat.

The role of glutamate receptors in processing noxious sensory input from the cornea was assessed in barbiturate-anesthetized rats. Animals were treated with selective antagonists for N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor subtypes prior to application of mustard oil to the corneal surface. Neural activation was estimated from the number of neurons that produced Fos, the protein product of the immediate early gene, c-fos, as detected by immunocytochemistry. Fos-positive neurons were found at two distinct regions of the spinal trigeminal nucleus: the subnucleus interpolaris/caudalis transition and the subnucleus caudalis/upper cervical cord transition. The number of Fos-positive neurons was reduced dose-dependently by the competitive N-methyl-D-aspartate receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (0.08-8 nmol, i.c.v.), or by the non-N-methyl-D-aspartate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (2.5-250 nmol, i.c.v.). The greatest reduction in Fos-positive cells was seen at the subnucleus caudalis/upper cervical cord transition after blockade of either receptor subtype. Combined blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors did not cause a further reduction in the number of Fos-positive neurons than was seen after the highest dose of either antagonist alone. Peripheral or central administration of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester, had no effect on the number of Fos-positive neurons after corneal stimulation. These results suggest that corneal input to neurons at the subnucleus caudalis/upper cervical cord transition, and to a lesser extent, at the subnucleus interpolaris/subnucleus caudalis transition depends on excitatory amino acid transmission. Both N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate receptor subtypes, but not the formation of nitric oxide, contribute to the processing of acute corneal stimuli by central trigeminal neurons.

Central trigeminal effects of somatostatin and etorphine on adrenal and autonomic function in the cat.

The influence of somatostatin and the potent mu-opiate receptor agonist etorphine on adrenal and autonomic responses mediated by trigeminal neurons was examined in chloralose-anesthetized cats. Microinjections of somatostatin (100 pmol) into laminae I-II of trigeminal subnucleus caudalis (Vc) evoked increases in the adrenal secretion of catecholamines and adrenal blood flow without affecting arterial pressure, heart rate, or plasma adrenocorticotropic hormone. Injections of somatostatin into laminae III-IV of Vc had no effect. Microinjections of etorphine (500 pmol) into laminae I-II of Vc had no effect, whereas injections into laminae III-IV caused small increases in total adrenal blood flow and peripheral concentrations of norepinephrine. To assess local effects of these drugs on peripheral trigeminal nociceptor-evoked autonomic responses, corneal stimulation was presented before and after topical application of somatostatin or etorphine on the dorsal brain stem surface. Somatostatin did not affect the adrenal and autonomic responses evoked by noxious thermal or chemical irritant stimulation of the cornea. Topical etorphine blocked completely the increase in adrenal blood flow, arterial pressure, and heart rate after noxious thermal stimulation of the cornea. These results suggest that somatostatin and mu-opiate receptor agonists act by different mechanisms at the level of the spinal trigeminal complex to affect adrenal and autonomic function.

Wound fluid amino acid concentrations regulate the effect of epidermal growth factor on fibroblast replication.

Growth factors and amino acids (AA) are required for cell proliferation. A comparison of the AA composition of wound fluid (WF) to that of Eagle's medium reveals that AA in WF may be limiting to cell replication. Yet WF supports fibroblast replication and stimulates AA uptake. Epidermal growth factor (EGF) stimulates fibroblast replication and stimulates human wound healing when applied topically. We evaluated the interactions between EGF and AA concentrations found in WF. Wound fibroblasts were cultured in media prepared to mimic the AA concentrations found in WF on days 1, 5, and 10 and in the presence of varying concentrations of EGF. Fibroblasts cultured in all three experimental media showed a dose response to EGF for both tritiated-thymidine uptake (proliferation) and AA uptake. The fibroblast proliferation in response to EGF was augmented by the AA composition of day-5 WF. These data show a dose-dependent effect of EGF on fibroblast replication and AA uptake in the absence of serum that is augmented by the particular AA combination found in day-5 WF and suggests that an optimal physiologic AA profile may aid in EGF stimulation of wound fibroblast replication.

Sympathetic control of adrenal secretion of enkephalins after hemorrhage in awake dogs.

Adrenomedullary secretion of methionine (Met)-enkephalin and of leucine (Leu)-enkephalin was measured in awake trained dogs subjected to 10 or 20% hemorrhage. Enkephalin secretory rates increased in proportion to the magnitude of hemorrhage with the ratio of Met-enkephalin to Leu-enkephalin being approximately 4:1. Arterial concentrations of enkephalins reflected adrenal secretion only in dogs exhibiting the largest secretory responses to hemorrhage. Cross-correlation analysis of 12 individual experiments showed that Met-enkephalin and epinephrine secretory rates were maximally correlated at 0-min delay, suggesting that enkephalins are cosecreted with catecholamines. The molar ratio in adrenal venous blood of epinephrine to Met-enkephalin ranges from 480 +/- 50 to 570 +/- 60. Chronic denervation of the adrenal by removing thoracic and lumbar splanchnic nerves prevented the Met-enkephalin and catecholamine secretory responses to hemorrhage. These data show that the adrenal medulla secretes enkephalins in response to stimuli activated by hemorrhage. These findings suggest that secretion of opiate-like peptides into the peripheral circulation may be a component of the sympathetic response to trauma.