Functional activation of the periaqueductal gray matter during conditioned and unconditioned fear in guinea pigs confronted with the Boa constrictor constrictor snake.

The periaqueductal gray matter (PAG) is an essential structure involved in the elaboration of defensive responses, such as when facing predators and conspecific aggressors. Using a prey vs predator paradigm, we aimed to evaluate the PAG activation pattern evoked by unconditioned and conditioned fear situations. Adult male guinea pigs were confronted either by a Boa constrictor constrictor wild snake or by the aversive experimental context. After the behavioral test, the rodents were euthanized and the brain prepared for immunohistochemistry for Fos protein identification in different PAG columns. Although Fos-protein-labeled neurons were found in different PAG columns after both unconditioned and conditioned fear situations at the caudal level of the PAG, we found greater activation of the lateral column compared to the ventrolateral and dorsomedial columns after predator exposure. Moreover, the lateral column of the PAG showed higher Fos-labeled cells at the caudal level compared to the same area at the rostral level. The present results suggested that there are different activation patterns of PAG columns during unconditioned and conditioned fear in guinea pigs. It is possible to hypothesize that the recruitment of specific PAG columns depended on the nature of the threatening stimulus.

Functional activation of the periaqueductal gray matter during conditioned and unconditioned fear in guinea pigs confronted with the Boa constrictor constrictor snake

The periaqueductal gray matter (PAG) is an essential structure involved in the elaboration of defensive responses, such as when facing predators and conspecific aggressors. Using a prey vs predator paradigm, we aimed to evaluate the PAG activation pattern evoked by unconditioned and conditioned fear situations. Adult male guinea pigs were confronted either by a Boa constrictor constrictor wild snake or by the aversive experimental context. After the behavioral test, the rodents were euthanized and the brain prepared for immunohistochemistry for Fos protein identification in different PAG columns. Although Fos-protein-labeled neurons were found in different PAG columns after both unconditioned and conditioned fear situations at the caudal level of the PAG, we found greater activation of the lateral column compared to the ventrolateral and dorsomedial columns after predator exposure. Moreover, the lateral column of the PAG showed higher Fos-labeled cells at the caudal level compared to the same area at the rostral level. The present results suggested that there are different activation patterns of PAG columns during unconditioned and conditioned fear in guinea pigs. It is possible to hypothesize that the recruitment of specific PAG columns depended on the nature of the threatening stimulus.

Temporomandibular inflammation mobilizes parvalbumin and FosB/deltaFosB neurons of amygdala and dorsal raphe.

Pathophysiological mechanisms involved in orofacial pain and their relationship with emotional disorders have emerged as an important research area for multidisciplinary studies. In particular, temporomandibular disorders (TMD) have been evaluated clinically from both physiological and psychological perspectives. We hypothesized that an altered neuronal activity occurs in the amygdala and the dorsal raphe nucleus (DR), encephalic regions involved in the modulation of painful and emotional information. Adult male Wistar rats were used in an experimental complete Freund's adjuvant (CFA)-induced temporomandibular joint (TMJ) inflammation model. CFA was applied for 1 or 10 days, and the animals were euthanized for brain samples dissection for FosB/ΔFosB and parvalbumin (PV) immunostaining. Our results were consistent in showing that the amygdala and DR were activated in the persistent inflammatory phase (10 days) and that the expression of PV+ interneurons in the amygdala was decreased. In contrast, in the DR, the expression of PV+ interneurons was increased in persistent states of CFA-induced TMJ inflammation. Moreover, at 10 days of inflammation, there was an increased co-localization of PV+ and FosB/ΔFosB+ neurons in the basolateral and central nucleus of the amygdala. Different nuclei of the amygdala, as well as portions of the DR, were activated in the persistent phase (10 days) of TMJ inflammation. In conclusion, altered activity of the amygdala and DR was detected during persistent inflammatory nociception in the temporomandibular joint. These regions may be essential for both sensory and affective dimensions of orofacial pain.

Temporomandibular inflammation mobilizes parvalbumin and FosB/deltaFosB neurons of amygdala and dorsal raphe

Pathophysiological mechanisms involved in orofacial pain and their relationship with emotional disorders have emerged as an important research area for multidisciplinary studies. In particular, temporomandibular disorders (TMD) have been evaluated clinically from both physiological and psychological perspectives. We hypothesized that an altered neuronal activity occurs in the amygdala and the dorsal raphe nucleus (DR), encephalic regions involved in the modulation of painful and emotional information. Adult male Wistar rats were used in an experimental complete Freund's adjuvant (CFA)-induced temporomandibular joint (TMJ) inflammation model. CFA was applied for 1 or 10 days, and the animals were euthanized for brain samples dissection for FosB/ΔFosB and parvalbumin (PV) immunostaining. Our results were consistent in showing that the amygdala and DR were activated in the persistent inflammatory phase (10 days) and that the expression of PV+ interneurons in the amygdala was decreased. In contrast, in the DR, the expression of PV+ interneurons was increased in persistent states of CFA-induced TMJ inflammation. Moreover, at 10 days of inflammation, there was an increased co-localization of PV+ and FosB/ΔFosB+ neurons in the basolateral and central nucleus of the amygdala. Different nuclei of the amygdala, as well as portions of the DR, were activated in the persistent phase (10 days) of TMJ inflammation. In conclusion, altered activity of the amygdala and DR was detected during persistent inflammatory nociception in the temporomandibular joint. These regions may be essential for both sensory and affective dimensions of orofacial pain.

Nociceptive Response to L-DOPA-Induced Dyskinesia in Hemiparkinsonian Rats.

Non-motor symptoms are increasingly identified to present clinical and diagnostic importance for Parkinson's disease (PD). The multifactorial origin of pain in PD makes this symptom of great complexity. The dopamine precursor, L-DOPA (L-3,4-dihydroxyphenylalanine), the classic therapy for PD, seems to be effective in pain threshold; however, there are no studies correlating L-DOPA-induced dyskinesia (LID) and nociception development in experimental Parkinsonism. Here, we first investigated nociceptive responses in a 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease to a hind paw-induced persistent inflammation. Further, the effect of L-DOPA on nociception behavior at different times of treatment was investigated. Pain threshold was determined using von Frey and Hot Plate/Tail Flick tests. Dyskinesia was measured by abnormal involuntary movements (AIMs) induced by L-DOPA administration. This data is consistent to show that 6-OHDA-lesioned rats had reduced nociceptive thresholds compared to non-lesioned rats. Additionally, when these rats were exposed to a persistent inflammatory challenge, we observed increased hypernociceptive responses, namely hyperalgesia. L-DOPA treatment alleviated pain responses on days 1 and 7 of treatment, but not on day 15. During that period, we observed an inverse relationship between LID and nociception threshold in these rats, with a high LID rate corresponding to a reduced nociception threshold. Interestingly, pain responses resulting from CFA-induced inflammation were significantly enhanced during established dyskinesia. These data suggest a pro-algesic effect of L-DOPA-induced dyskinesia, which is confirmed by the correlation founded here between AIMs and nociceptive indexes. In conclusion, our results are consistent with the notion that central dopaminergic mechanism is directly involved in nociceptive responses in Parkinsonism condition.

Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats.

The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats.

Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats

The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats.

Laser therapy reduces gelatinolytic activity in the rat trigeminal ganglion during temporomandibular joint inflammation.

OBJECTIVES: To investigate whether low-level laser therapy (LLLT) alters the expression and activity of MMP-2 and MMP-9 in the trigeminal ganglion (TG) during different stages of temporomandibular joint (TMJ) inflammation in rats. It also evaluated whether LLLT modifies mechanical allodynia and orofacial hyperalgesia. MATERIALS AND METHODS: Wistar rats (±250 g) were divided into groups that received saline (SAL) or complete Freund's adjuvant (CFA, 50 µl) in the TMJ, and that later underwent LLLT (20 J cm(-2) ) at their TMJ or not (groups SAL, SAL + LLLT, CFA, and CFA + LLLT). LLLT was applied on days 3, 5, 7, and 9 after SAL or CFA. Mechanical allodynia was evaluated on days 1, 3, 5, 7, and 10; orofacial hyperalgesia was assessed on day 10. Gelatin zymography and in situ zymography aided quantification of MMPs in the TG. RESULTS: Low-level laser therapy abolished the reduction in the mechanical orofacial threshold and the increase in orofacial rubbing during the orofacial formalin test induced by CFA. LLLT also decreased the CFA-induced rise in the levels of MMP-9 and MMP-2 as well as the gelatinolytic activity in the TG. CONCLUSION: Low-level laser therapy could constitute an adjuvant therapy to treat temporomandibular disorders and prevent inflammation-induced alterations in the levels of MMP-2 and MMP-9 and in the gelatinolytic activity in TGs.

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent.

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3',5'-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

Expression of MMP-2 and MMP-9 in the rat trigeminal ganglion during the development of temporomandibular joint inflammation.

Orofacial pain is a prevalent symptom in modern society. Some musculoskeletal orofacial pain is caused by temporomandibular disorders (TMDs). This condition has a multi-factorial etiology, including emotional factors and alteration of the masticator muscle and temporomandibular joints (TMJs). TMJ inflammation is considered to be a cause of pain in patients with TMD. Extracellular proteolytic enzymes, specifically the matrix metalloproteinases (MMPs), have been shown to modulate inflammation and pain. The purpose of this investigation was to determine whether the expression and level of gelatinolytic activity of MMP-2 and MMP-9 in the trigeminal ganglion are altered during different stages of temporomandibular inflammation, as determined by gelatin zymography. This study also evaluated whether mechanical allodynia and orofacial hyperalgesia, induced by the injection of complete Freund's adjuvant into the TMJ capsule, were altered by an MMP inhibitor (doxycycline, DOX). TMJ inflammation was measured by plasma extravasation in the periarticular tissue (Evans blue test) and infiltration of polymorphonuclear neutrophils into the synovial fluid (myeloperoxidase enzyme quantification). MMP expression in the trigeminal ganglion was shown to vary during the phases of the inflammatory process. MMP-9 regulated the early phase and MMP-2 participated in the late phase of this process. Furthermore, increases in plasma extravasation in periarticular tissue and myeloperoxidase activity in the joint tissue, which occurred throughout the inflammation process, were diminished by treatment with DOX, a nonspecific MMP inhibitor. Additionally, the increases of mechanical allodynia and orofacial hyperalgesia were attenuated by the same treatment.

Expression of MMP-2 and MMP-9 in the rat trigeminal ganglion during the development of temporomandibular joint inflammation

Orofacial pain is a prevalent symptom in modern society. Some musculoskeletal orofacial pain is caused by temporomandibular disorders (TMDs). This condition has a multi-factorial etiology, including emotional factors and alteration of the masticator muscle and temporomandibular joints (TMJs). TMJ inflammation is considered to be a cause of pain in patients with TMD. Extracellular proteolytic enzymes, specifically the matrix metalloproteinases (MMPs), have been shown to modulate inflammation and pain. The purpose of this investigation was to determine whether the expression and level of gelatinolytic activity of MMP-2 and MMP-9 in the trigeminal ganglion are altered during different stages of temporomandibular inflammation, as determined by gelatin zymography. This study also evaluated whether mechanical allodynia and orofacial hyperalgesia, induced by the injection of complete Freund's adjuvant into the TMJ capsule, were altered by an MMP inhibitor (doxycycline, DOX). TMJ inflammation was measured by plasma extravasation in the periarticular tissue (Evans blue test) and infiltration of polymorphonuclear neutrophils into the synovial fluid (myeloperoxidase enzyme quantification). MMP expression in the trigeminal ganglion was shown to vary during the phases of the inflammatory process. MMP-9 regulated the early phase and MMP-2 participated in the late phase of this process. Furthermore, increases in plasma extravasation in periarticular tissue and myeloperoxidase activity in the joint tissue, which occurred throughout the inflammation process, were diminished by treatment with DOX, a nonspecific MMP inhibitor. Additionally, the increases of mechanical allodynia and orofacial hyperalgesia were attenuated by the same treatment.

Effects of short-term acetaminophen and celecoxib treatment on orthodontic tooth movement and neuronal activation in rat.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been used for pain relief in orthodontics, but clinical studies reported that they may reduce tooth movement (TM). By other side, TM seems to activate brain structures related to nociception, but the effects of NSAIDs in this activation have not been studied yet. We analyzed the effect of short-term treatment with acetaminophen or celecoxib in the separation of rat upper incisors, as well as in neuronal activation of the spinal trigeminal nucleus, following tooth movement. Thirty rats (400-420 g) were pretreated through oral gavage (1 ml/dose) with acetaminophen (200mg/kg), celecoxib (50mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance for TM. In controls, this appliance was immediately removed after its introduction. Rats received ground food, and every 12h, one of the drugs or vehicle. After 48 h, they were anesthetized, maxilla was radiographed, and were perfused with 4% paraformaldehyde. Brains were further processed for Fos immunohistochemistry. TM induced incisor distalization (p<0.05) and neuronal activation of the spinal trigeminal nucleus. Treatment with both drugs did not affect tooth movement, but reduced c-fos expression in the caudalis subnucleus. No changes in c-fos expression were seen in the oralis and interpolaris subnuclei. We conclude that neither celecoxib nor acetaminophen seems to affect tooth movement, when used for 2 days, but both drugs are able to reduce the activation of brain structures related to nociception. Short-term treatment with celecoxib, thus, may be a therapeutic alternative to acetaminophen when the latter is contraindicated.

Rapid maxillary expansion causes neuronal activation in brain structures of rats.

A correlation between pain sensation and neuronal c-fos expression has been analyzed following experimental rapid maxillar expansion (RME). Adult male Wistar rats were anaesthetized and divided into three groups: animals that received an orthodontic apparatus, which was immediately removed after the insertion (control), animals that received an inactivated orthodontic apparatus (without force), and animals that received an orthodontic apparatus previously activated (140 g force). After 6, 24, 48, or 72 h, the animals were re-anaesthetized, and perfused with 4% paraformaldehyde. The brains were removed, fixed, and sections containing brain structures related to nociception were processed for Fos protein immunohistochemistry (IHC). The insertion of the orthodontic apparatus with 140 g was able to cause RME that could be seen by radiography. The IHC results showed that the number of activated neurons in the different nuclei changed according to the duration of appliance insertion and followed a temporal pattern similar to that of sensations described in clinics. The animals that received the orthodontic apparatus without force did not show RME but a smaller c-fos expression in the same brain structures. In conclusion, we demonstrate that orthodontic force used for palate disjunction activates brain structures that are related to nociception, and that this activation is related to the pain sensation described during orthodontic treatment.

Cholinergic-opioidergic interaction in the central amygdala induces antinociception in the guinea pig

Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.

Cholinergic-opioidergic interaction in the central amygdala induces antinociception in the guinea pig.

Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.

Endogenous opiate analgesia induced by tonic immobility in guinea pigs

A function of the endogenous analgesic system is to prevent recuperative behaviors generated by tissue damage, thus preventing the emission of species-specific defensive behaviors. Activation of intrinsic nociception is fundamental for the maintenance of the behavioral strategy adopted. Tonic immobility (TI) is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. We studied the effect of TI behavior on nociception produced by the formalin and hot-plate tests in guinea pigs. The induction of TI produced a significant decrease in the number of flinches (18 + or - 6 and 2 + or - 1 in phases 1 and 2) and lickings (6 + or - 2 and 1 + or - 1 in phases 1 and 2) in the formalin test when compared with control (75 + or - 13 and 22 + or - 6 flinches in phases 1 and 2; 28 + or - 7 and 17 + or - 7 lickings in phases 1 and 2). In the hot-plate test our results also showed antinociceptive effects of TI, with an increase in the index of analgesia 30 and 45 min after the induction of TI (0.67 0.1 and 0.53 + or - 0.13, respectively) when compared with control (-0.10 + or - 0.08 at 30 min and -0.09 0.09 at 45 min). These effects were reversed by pretreatment with naloxone (1 mg/kg, ip), suggesting that the hypoalgesia observed after induction of TI behavior, as evaluated by the algesimetric formalin and hot-plate tests, is due to activation of endogenous analgesic mechanisms involving opioid synapses