Effects of aging on basement membrane of tibialis anterior muscle during recovery following muscle injury in rats.

We investigated the effect of aging on the basement membrane (BM) during postinjury muscle recovery. Using a rat model, we found that aging delayed muscle fiber and BM recovery. In addition, expression of BM-related factors peaked 7 days after muscle injury among both young and older rats. Peak expression of collagen IV synthetic factors decreased with age, whereas expression of the degradative factor was unaffected by age. These results suggest that age-related delays in postinjury muscle fiber and BM recovery may be related to the suppression of collagen IV synthetic factors.

Effects of Endurance Exercise on Basement Membrane in the Soleus Muscle of Aged Rats.

The basement membrane (BM)-related factors, including collagen IV, are important for the maintenance and recovery of skeletal muscles. Aging impairs the expression of BM-related factors during recovery after disuse atrophy. Muscle activity facilitates collagen synthesis that constitutes the BM. However, the effect of endurance exercise on the BM of aged muscles is unclear. Thus, to understand the effect of endurance exercise on the BM of the skeletal muscle in aged rats, we prescribed treadmill running in aged rats and compared the differences in the expression of BM-related factors between the aged rats with and without exercise habits. Aged rats were subjected to endurance exercise via treadmill running. Exercise increased the mRNA expression levels of the BM-related factors, the area and intensity of collagen IV-immunoreactivity and the width of lamina densa in the soleus muscle of aged rats. These finding suggests that endurance exercise promotes BM construction in aged rats.

Effects of aging on basement membrane-related gene expression of the skeletal muscle in rats.

The basement membrane (BM), with collagen IV as a major component, plays an important role in the maintenance of muscle structure and its robustness. To investigate the effects of aging on factors related to BM construction, we compared the expression status of these factors in 3- and 20-month-old male Wistar rats. The expression levels of Col4a1 and Col4a2 (encoding collagen IV), Sparc (involved in collagen IV functionalization), and Mmp14 (a collagen IV degradation factor) were decreased. These results suggest that aging suppresses collagen IV synthetic and degradative factors and affects BM-related factors in the steady state.

Basement membrane recovery process in rat soleus muscle after exercise-induced muscle injury.

Purpose: Collagen IV is a component of the basement membrane (BM) that provides mechanical support for muscle fibers. In addition, transcription factor 4 (TCF4) is highly expressed in muscle connective tissue fibroblasts and regulates muscle regeneration. However, the expression of collagen IV and TCF4 (+) cells in response to exercise-induced muscle injury is not well known. Here, we investigated the expression and localization of collagen IV and TCF4 (+) cells during the recovery process after muscle injury induced by different exercise loads.Materials and Methods: Muscle injury was observed in the soleus muscle of young Wistar rats after 12 or 18 sets-downhill running (DR) on a treadmill. After running, the rats were permitted to recover for a period of 0.5 days, 2 days, or 7 days.Results: Ectopic localization of collagen IV in injured muscle fibers was observed after DR, and the number increased at 0.5 days after 18 sets DR and at 2 days after 12 or 18 sets DR as compared to the number observed at baseline. BM disruption was observed after DR. TCF4 (+) cells appeared in the inside and around injured muscle fibers at 0.5 day of recovery. After 18 sets DR, TCF4 (+) cells were more abundant for a longer period than that observed after 12 sets DR.Conclusions: DR induces BM disruption accompanied by muscle fiber damage. It is possible that BM destruction may be accompanied by muscle damage and that TCF4 (+) cells contribute to muscle fiber and BM recovery.

Effects of aging on basement membrane of the soleus muscle during recovery following disuse atrophy in rats.

Aging is known to lead to the impaired recovery of muscle after disuse as well as the increased susceptibility of the muscle to damage. Here, we show that, in the older rats, reloading after disuse atrophy, causes the damage of the muscle fibers and the basement membrane (BM) that structurally support the muscle fibers. Male Wistar rats of 3-(young) and 20-(older) months of age were subjected to hindlimb-unloading for 2weeks followed by reloading for a week. In the older rats, the soleus muscles showed necrosis and central nuclei fiber indicating the regeneration of muscle fibers. Furthermore, ectopic immunoreactivity of collagen IV, a major component of the BM, remained mostly associated with the necrotic appearance, suggesting that the older rats were impaired with the ability of repairing the damaged BM. Further, after unloading and reloading, the older rats did not show a significant alteration, although the young rats showed clear response of Col4a1 and Col4a2 genes, both coding for collagen IV. In addition, during the recovery phase, the young rats showed increase in the amount of Hsp47 and Sparc mRNA, which are protein folding-related factor genes, while the older rats did not show any significant variation. Taken together, our findings suggest that the atrophic muscle fibers of the older rats induced by unloading were vulnerable to the weight loading, and that attenuated reactivity of the BM-synthesizing fibroblast to gravity contributes to the fragility of muscle fibers in the older animals.

Selective elimination of isolectin B4-binding trigeminal neurons enhanced formalin-induced nocifensive behavior in the upper lip of rats and c-Fos expression in the trigeminal subnucleus caudalis.

The functional significance of non-peptidergic C-fibers in orofacial pain processing is largely unknown. The present study examined the effects of the selective elimination of isolectin B4 (IB4)-binding (IB4(+)) neurons on formalin-induced face rubbing behavior (FRB) in the upper lip of rats and c-Fos-immunoreactive (c-Fos-IR) cells in the trigeminal subnucleus caudalis (Vc). IB4 conjugated to neurotoxin, saporin (IB4-Sap), blank-saporin (Bl-Sap), or saline (Sal) was injected into the cisterna magna. IB4-Sap treatments significantly decreased IB4(+) terminals in lamina II of Vc and IB4(+) trigeminal ganglia neurons, whereas Sal- and BI-Sap treatments did not. The number of formalin-induced FRB 15-30 min after the formalin injection was significantly higher in IB4-Sap-treated rats than in Sal- or Bl-Sap-treated rats, and was associated with an increase in c-Fos-IR cells. The systemic preadministration of the GABAA antagonist, bicuculline, and agonist, muscimol, had stronger decreasing effects on FRB and c-Fos-IR cells in IB4-Sap-treated rats than the preadministration of Sal, whereas the opposite effects were observed in Sal- and Bl-Sap-treated rats. These results indicate that IB4(+) neurons in the trigeminal nerve play antinociceptive regulatory roles in formalin-induced orofacial pain processing and that GABAA receptor functions at segmental and supratrigeminal sites have complex modulatory influences on antinociceptive roles.

Inferior alveolar nerve transection enhanced formalin-induced nocifensive responses in the upper lip: systemic buprenorphine had more antinociceptive efficacy over morphine.

This study was designed to investigate the efficacy of a partial µ-opioid agonist, buprenorphine, against the formalin-induced hyperalgesia in the upper lip in chronically inferior alveolar nerve (IAN)-transected rats. Subcutaneous injection of diluted formalin into the upper lip in the IAN-transected rats showed an increased number of pain-related behavior (PRB; face-rubbing behavior) in every phase up to 45 min (p < 0.01) compared with that in the nontransected sham control rats. The numbers of c-Fos-immunoreactive (IR) cells in the superficial layers of the trigeminal nucleus caudalis (VcI/II) at the rostral (0-0.7 mm caudal to the obex) and middle levels (1.4-2.2 mm caudal to the obex) 2 h after the formalin injection in the IAN-transected rats were significantly increased compared with those in the control rats. The PRB in phases 1 and 2 (0-15 and 15-30 min after formalin injection) in rats with preadministration of morphine (3 mg/kg i.p.) or buprenorphine (100 µg/kg i.p.) was significantly (p < 0.05) smaller than those in the control rats. There was no significant difference in the efficacy between morphine and buprenorphine at these doses. The antinociceptive efficacy in phase 2 of buprenorphine (100 µg/kg) was higher (p < 0.05) than that of morphine (3 mg/kg) in the IAN-transected rats. The number of c-Fos-IR cells in the VcI/II at every level (0-3.6 mm caudal to the obex) after formalin injection was significantly decreased (p < 0.01) with preadministration of morphine (3 mg/kg) or buprenorphine (100 µg/kg) in the control rats. In the IAN-transected rats, the number of c-Fos-IR cells in the caudal VcI/II (2.2-3.6 mm caudal to the obex) after formalin injection was significantly decreased (p < 0.01) with preadministration of buprenorphine (100 µg/kg) but not so much (2.2-2.9 mm caudal to the obex, p < 0.05; 2.9-3.6 mm caudal to the obex, p > 0.05) with preadministration of morphine (3 mg/kg). These results indicate that IAN transection enhanced formalin-induced nocifensive responses in the upper lip, the dermatome of the intact nerve neighboring the IAN. Systemic preadministration of buprenorphine had more antinociceptive effects on the formalin-induced nocifensive behavior in the upper lip compared with morphine in the IAN-transected rats.

Mechanisms of orofacial pain control in the central nervous system.

Recent advances in the study of pain have revealed somatotopic- and modality-dependent processing and the integration of nociceptive signals in the brain and spinal cord. This review summarizes the uniqueness of the trigeminal sensory nucleus (TSN) in structure and function as it relates to orofacial pain control. The oral nociceptive signal is primarily processed in the rostral TSN above the obex, the nucleus principalis (Vp), and the subnuclei oralis (SpVo) and interpolaris (SpVi), while secondarily processed in the subnucleus caudalis (SpVc). In contrast, the facial nociceptive signal is primarily processed in the SpVc. The neurons projecting to the thalamus are localized mostly in the Vp, moderately in the SpVi, and modestly in the ventrolateral SpVo and the SpVc. Orofacial sensory inputs are modulated in many different ways: by interneurons in the TSN proper, through reciprocal connection between the TSN and rostral ventromedial medulla, and by the cerebral cortex. A wide variety of neuroactive substances, including substance P, gamma-aminobutyric acid, serotonin and nitric oxide (NO) could be involved in the modulatory functions of these curcuits. The earliest expression of NO synthase (NOS) in the developing rat brain is observed in a discrete neuronal population in the SpVo at embryonic day 15. NOS expression in the SpVc is late at postnatal day 10. The neurons receiving intraoral signals are intimately related with the sensorimotor reflexive function through the SpVo. In summary, a better understanding of the trigeminal sensory system--which differs from the spinal system--will help to find potential therapeutic targets and lend to developing new analgesics for orofacial-specific pain with high efficacy and fewer side effects.

Demonstration of a trigeminothalamic pathway to the oval paracentral intralaminar thalamic nucleus and its involvement in the processing of noxious orofacial deep inputs.

Using combined retrograde labeling and Fos protein immunohistochemistry, we show that after masseter inflammation, a population of neurons in the dorsal portion of the subnuclei interpolaris/caudalis transition zone at the level of the obex was activated and projected to the oval paracentral nucleus (OPC) of the intralaminar thalamic nuclei. The present findings indicate a trigeminothalamic pathway to the OPC intralaminar nucleus involved in central processing of orofacial deep noxious input.

Trigeminal transition zone/rostral ventromedial medulla connections and facilitation of orofacial hyperalgesia after masseter inflammation in rats.

Recent studies have implicated a role for the trigeminal interpolaris/caudalis (Vi/Vc) transition zone in response to orofacial injury. Using combined neuronal tracing and Fos protein immunocytochemistry, we investigated functional connections between the Vi/Vc transition zone and rostral ventromedial medulla (RVM), a key structure in descending pain modulation. Rats were injected with a retrograde tracer, FluoroGold, into the RVM 7 days before injection of an inflammatory agent, complete Freund's adjuvant, into the masseter muscle and perfused at 2 hours postinflammation. A population of neurons in the ventral Vi/Vc overlapping with caudal ventrolateral medulla, and lamina V of the trigeminal subnucleus caudalis (Vc), exhibited FluoroGold/Fos double staining, suggesting the activation of the trigeminal-RVM pathway after inflammation. No double-labeled neurons were found in the dorsal Vi/Vc and laminae I-IV of Vc. Injection of an anterograde tracer, Phaseolus vulgaris leucoagglutinin, into the RVM resulted in labeling profiles overlapped with the region that showed FluoroGold/Fos double labeling, suggesting reciprocal connections between RVM and Vi/Vc. Lesions of Vc with a soma-selective neurotoxin, ibotenic acid, significantly reduced inflammation-induced Fos expression as well as the number of FluoroGold/Fos double-labeled neurons in the ventral Vi/Vc (P<0.05). Compared with control rats, lesions of the RVM (n=6) or Vi/Vc (n=6) with ibotenic acid led to the elimination or attenuation of masseter hyperalgesia/allodynia developed after masseter inflammation (P<0.05-0.01). The present study demonstrates reciprocal connections between the ventral Vi/Vc transition zone and RVM. The Vi/Vc-RVM pathway is activated after orofacial deep tissue injury and plays a critical role in facilitating orofacial hyperalgesia.

Antibody array analysis of peripheral and blood cytokine levels in rats after masseter inflammation.

This study was undertaken to evaluate the changes in cytokine levels in response to orofacial deep tissue inflammation. Inflammation was induced by injecting complete Freund's adjuvant (CFA, 0.05 ml 1:1 oil/saline suspension) into the masseter of the male Sprague-Dawley rat under brief halothane anesthesia. At 30 min, 5 h and 24 h after CFA injection (n = 3-4/time point), tissues were dissected from masseter and total proteins isolated. Rat Cytokine Antibody Array 1.1 (RayBiotech) coated with 19 specific cytokine antibodies were probed with protein samples and the relative cytokine levels were compared. Compared to saline-injected rats, there were significant increases (p < 0.05-0.01) in the levels of seven cytokines in the masseter tissue after CFA, including interleukin (IL)-1beta (5 h), IL-6 (5 h), tumor necrosis factor-alpha (5 h), monocyte chemoattractant protein-1 (5 h, 24 h), cytokine-induced neutrophil chemoattractant-2 and -3 (5 h, 24 h), and tissue inhibitor of metalloproteinase-1 (5 h, 24 h). All 19 cytokines were detected in the blood samples, but they did not show significant changes after inflammation. Masseter hyperalgesia and allodynia occurred at 30 min and persisted at 5-24 h after inflammation, as assessed by probing the skin above the masseter with von Frey filaments. The present results indicate selective localized cytokine responses to masseter inflammation. Although different cytokines exist in the blood, their levels did not mirror, nor did not appear to depend on, the local cytokine levels. The findings provide specific targets for further studying the involvement of cytokines in orofacial inflammation and hyperalgesia.

Bilateral projection of functionally characterized trigeminal oralis neurons to trigeminal motoneurons in cats.

Intracellular Neurobiotin-injections were used to label functionally identified neurons in the rostro-dorsomedial part of the trigeminal oral nucleus (Vo.r) in the cat. The labeled Vo.r neurons with the mechanoreceptive field in oral tissues innervated bilaterally either jaw-opening motoneurons or jaw-closing motoneurons. This result suggests that Vo.r neurons play an important role in sensory-motor reflexes responsible for coordination of bilaterally symmetrical jaw movements.

Group I metabotropic glutamate receptor NMDA receptor coupling and signaling cascade mediate spinal dorsal horn NMDA receptor 2B tyrosine phosphorylation associated with inflammatory hyperalgesia.

Hindpaw inflammation induces tyrosine phosphorylation (tyr-P) of the NMDA receptor (NMDAR) 2B (NR2B) subunit in the rat spinal dorsal horn that is closely related to the initiation and development of hyperalgesia. Here, we show that in rats with Freund's adjuvant-induced inflammation, the increased dorsal horn NR2B tyr-P is blocked by group I metabotropic glutamate receptor (mGluR) antagonists [7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP), by the Src inhibitor CGP 77675, but not by the MAP kinase inhibitor 2'-amino-3'-methoxyflavone. Analysis of the calcium pathways shows that the in vivo NR2B tyr-P is blocked by an IP3 receptor antagonist 2-aminoethoxydiphenylborate (2APB) but not by antagonists of ionotropic glutamate receptors and voltage-dependent calcium channels, suggesting that the NR2B tyr-P is dependent on intracellular calcium release. In a dorsal horn slice preparation, the group I (dihydroxyphenylglycine), but not group II [(2R,4R)-4-aminopyrrolidine-2,3-dicarboxylate] and III [L-AP 4 (L-(+)-2-amino-4-phosphonobutyric acid)], mGluR agonists, an IP3 receptor (D-IP3) agonist, and a PKC (PMA) activator, induces NR2B tyr-P similar to that seen in vivo after inflammation. Coimmunoprecipitation indicates that Shank, a postsynaptic density protein associated with mGluRs, formed a complex involving PSD-95 (postsynaptic density-95), NR2B, and Src in the spinal dorsal horn. Double immunofluorescence studies indicated that NR1 is colocalized with mGluR5 in dorsal horn neurons. mGluR5 also coimmunoprecipitates with NR2B. Finally, intrathecal pretreatment of CPCCOEt, MPEP, and 2APB attenuates inflammatory hyperalgesia. Thus, inflammation and mGluR-induced NR2B tyr-P share similar mechanisms. The group ImGluR-NMDAR coupling cascade leads to phosphorylation of the NMDAR and appears necessary for the initiation of spinal dorsal horn sensitization and behavioral hyperalgesia after inflammation.

Differential rostral projections of caudal brainstem neurons receiving trigeminal input after masseter inflammation.

To understand the functional significance of orofacial injury-induced neuronal activation, this study examined the rostral projection of caudal brainstem neurons that were activated by masseteric inflammation. Rats were injected with a retrograde tracer, Fluorogold, into the nucleus submedius of the thalamus (Sm), parabrachial nucleus (PB), lateral hypothalamus (LH), or medial ventroposterior thalamic nucleus (VPM) 7 days before injection of an inflammatory agent, complete Freund's adjuvant (CFA), into the masseter muscle. Rats were perfused at 2 hours after inflammation, and brainstem tissues were processed for Fos-Fluorogold double immunocytochemistry. Although there was no difference in Fos expression among the four groups (n=4 per site), the rostral projection of Fos-positive neurons showed dramatic differences. In the ventral portion of the trigeminal subnuclei interpolaris/caudalis (Vi/Vc) transition zone, the percentage of Fos-positive neurons projecting to the Sm (39.7%) was significantly higher than that projecting to the LH (5.4%) or VPM (5.6%; P<.001). The anesthesia alone also induced Fos expression in ventral Vi/Vc neurons, but these neurons did not project to Sm. In the caudal laminated Vc and dorsal Vi/Vc, the PB was the major site of rostral projection of Fos-positive neurons. In the caudal ventrolateral medulla and nucleus tractus solitarius, Fos-positive neurons projected to the Sm, PB, and LH. Most VPM-projecting neurons examined did not show Fos-like immunoreactivity after masseter inflammation. These findings emphasize the importance of the trigeminal Vi/Vc transition zone in response to orofacial deep tissue injury. Furthermore, the results differentiate the ventral and dorsal portions of the Vi/Vc transition zone, in that the Sm received projection mainly from activated neurons in the ventral Vi/Vc. The activation of Vi/Vc neurons and associated ascending pathways may facilitate somatoautonomic and somatovisceral integration and descending pain modulation after orofacial deep tissue injury.

NADPH-diaphorase and calcium binding proteins in the trigeminal nucleus oralis of rats.

We have examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV), in the trigeminal nucleus oralis (Sp5O). NADPH-d was detected by histochemistry while CBP was detected by immunohistochemistry. NADPH-d-positive neurons were distributed in the medial rostro-dorsomedial part (RDMsp5O) and dorsomedial part (DMsp5O) of Sp5O, and the rostrolateral part of the nucleus of the solitary tract (NTS). CB- and CR-positive neurons were mainly distributed in the dorsal part of Sp5O. In contrast, PV-positive neurons were mainly distributed in the ventral part of Sp5O. NADPH-d colocalized with CB (40%) and CR (20%) but not with PV in neurons of DMsp5O/ NTS. The mean cell sizes of neurons in RDMsp5O were larger than those in DMsp5O/NTS. PV-positive neurons were larger than NADPH-d-positive neurons. NADPH-d-, CB- and CR-positive neurons were generally small in RDMsp5O and DMsp5O/NTS. Few neurons were retrogradely labeled in RDMsp5O and DMsp5O from the thalamus, when numerous labeled neurons were in the principal and interpolar nuclei. These data indicate that NADPH-d histochemistry and CB, CR and PV immunohistochemistry identify a discrete cell population in Sp5O. Those labeled neurons in RDMsp5O and DMsp5O/NTS were considered to be involved in sensorimotor reflexive function of the intra-oral structures.

Effects of somatosensory cortical stimulation on expression of c-Fos in rat medullary dorsal horn in response to formalin-induced noxious stimulation.

We examined the effects of epidural electrical stimulation of primary (SI) and secondary (SII) somatosensory cortex on expression of c-Fos protein in rat medullary dorsal horn neurons (Vc; trigeminal nucleus caudalis) in response to formalin-induced noxious stimulation. Epidural electrical stimulation (single pulse, 0.2 msec duration at 10 Hz) was applied to the left facial region SI or SII at three different stimulus intensities, 0.1, 0.5, and 1.0 mA for 60 min 0 or 2 hr after bilateral injection of formalin into the lower lip. SII stimulation at 1.0 mA immediately after injection of formalin, significantly decreased the number of Fos-positive cells in the right VcI/II by 32.4%. There was no significant change in the number of Fos-positive cells in the VcIII/IV. SII stimulation at 0.5 and 1.0 mA 2 hr after injection of formalin, significantly decreased the number of Fos-positive cells in the right VcI/II by 47.9% and 40.8%, but significantly increased the number of Fos-positive cells in the right VcIII/IV by 178.8% and 324.3%, respectively. In contrast, SI stimulation had no effect on expression of c-Fos in Vc. Possible direct corticotrigeminal projections were labeled anterogradely by injection of WGA-HRP into the SI and SII. In the Vc, labeled terminals were distributed mostly in the contralateral medial half of VcIII/IV and medullary reticular nucleus dorsalis but rarely in VcI/II. These results suggest that activation of SII-medullary fibers suppress nociceptive information from the oro-facial regions.