Effect of chronic lithium on mechanical sensitivity and trabecular bone loss induced by type-1 diabetes mellitus in mice.

Type-1 diabetes mellitus (T1DM) is a chronic condition characterized by long-term hyperglycemia that results in several complications such as painful peripheral neuropathy, bone deterioration, and increased risk of bone fractures. Lithium, a first-line therapy for bipolar disorder, has become an attractive agent for attenuating peripheral neuropathy and menopause-induced bone loss. Therefore, our aim was to determine the effect of chronic lithium treatment on mechanical hypersensitivity and trabecular bone loss induced by T1DM in mice. T1DM was induced in male C57BL/6J mice by intraperitoneal injection of streptozotocin (STZ, 50 mg/kg/day, for 5 consecutive days). 12 weeks after T1DM-induction, mice received a daily intraperitoneal injection of vehicle, 30 or 60 mg/kg lithium (as LiCl) for 6 weeks. Throughout the treatment period, blood glucose levels and mechanical sensitivity were evaluated every 2 weeks. After lithium treatment, the femur and L5 vertebra were harvested for microcomputed tomography (microCT) analysis. T1DM mice showed significant hyperglycemia, mechanical hypersensitivity, and significant trabecular bone loss as compared with the control group. Chronic lithium treatment did not revert the hindpaw mechanical hypersensitivity nor hyperglycemia associated to T1DM induced by STZ. In contrast, microCT analysis revealed that lithium reverted, in a dose-dependent manner, the loss of trabecular bone associated to T1DM induced by STZ at both the distal femur and L5 vertebra. Lithium treatment by itself did not affect any trabecular bone parameter in non-diabetic mice.

In Vivo Anti-Inflammatory Effect, Antioxidant Activity, and Polyphenolic Content of Extracts from Capsicum chinense By-Products.

By-products of Capsicum chinense Jacq., var Jaguar could be a source of bioactive compounds. Therefore, we evaluated the anti-inflammatory effect, antioxidant activity, and their relationship with the polyphenol content of extracts of habanero pepper by-products obtained from plants grown on black or red soils of Yucatán, Mexico. Moreover, the impact of the type of extraction on their activities was evaluated. The dry by-product extracts were obtained by maceration (ME), Soxhlet (SOX), and supercritical fluid extraction (SFE). Afterward, the in vivo anti-inflammatory effect (TPA-induced ear inflammation) and the in vitro antioxidant activity (ABTS) were evaluated. Finally, the polyphenolic content was quantified by Ultra-Performance Liquid Chromatography (UPLC), and its correlation with both bioactivities was analyzed. The results showed that the SFE extract of stems of plants grown on red soil yielded the highest anti-inflammatory effect (66.1 ± 3.1%), while the extracts obtained by ME and SOX had the highest antioxidant activity (2.80 ± 0.0052 mM Trolox equivalent) and polyphenol content (3280 ± 15.59 mg·100 g-1 dry basis), respectively. A negative correlation between the anti-inflammatory effect, the antioxidant activity, and the polyphenolic content was found. Overall, the present study proposed C. chinense by-products as a valuable source of compounds with anti-inflammatory effect and antioxidant activity.

000Synthesis of new α-aminophosphonates: Evaluation as anti-inflammatory agents and QSAR studies.

In this paper, we report the synthesis of a new series of α-aminophosphonates derivatives based in an efficient three-component reaction. All compounds prepared showed significant anti-inflammatory activity, being the compounds 1a, 1c, 1d, 1f, 2b and 2c the most promising ones, in terms of maximal efficacy (over 95%), potency (ED50 range between 0.7 and 10.1 mg/ear) and relative potency (range from 0.04 to 0.67). Compounds 1a, 1c, 1d and 1f significantly decrease the number of neutrophils (range from 46.7 to 63.0%) and monocytes (18.9-34.1%) in blood samples from the orbital sinus. Additionally, QSAR model revealed that the spherical molecular shape and the location of the HOMO on the phenyl ring improves the anti-inflammatory activity of the compounds. The values of R2, Q2, s and F statistical parameters and the QUIK, asymptotic Q2 and Overfitting rules validate the descriptive and predictive ability of the QSAR model. Altogether these results suggest that these new α-aminophosphonates are potential agents for the treatment of inflammation.

Protein hydrolysates and ultrafiltered < 1 KDa fractions from Phaseolus lunatus, Phaseolus vulgaris and Mucuna pruriens exhibit antihyperglycemic activity, intestinal glucose absorption and α-glucosidase inhibition with no acute toxicity in rodents.

BACKGROUND: Protein hydrolysates from food plants, such as legumes, have emerged as a new alternative to treat hyperglycemia, an important risk factor contributing to the development of type 2 diabetes mellitus (T2DM) and its complications. The aim of this work was to assess the antihyperglycemic activity and inhibition of α-glucosidase, and intestinal glucose absorption, and acute toxicity of total hydrolysates and < 1 kDa fractions from Phaseolus lunatus L., Phaseolus vulgaris L., and Mucuna pruriens (L.) DC., obtained by hydrolysis with Alcalase®-Flavourzyme® or pepsine-pancreatin enzymatic systems. RESULTS: In vivo results showed that three of six total hydrolysates and four of six < 1 kDa fractions suppressed starch-induced postprandial hyperglycemia (ED50 range between 1.4 and 93 mg kg-1 ). In vitro, total hydrolysates and fractions, particularly from M. pruriens, inhibited carbohydrate intestinal absorption (from 19.2 to 40%), and α-glucosidase activity (IC50 from 0.86 to 75 mg mL-1 ). Finally, none of the hydrolysates and fractions tested did not show any signs of toxicity (LD50 > 5000 mg kg-1 ). CONCLUSION: These results suggest that hydrolysates and < 1 kDa fractions from P. lunatus, P. vulgaris and M. pruriens are suitable candidates to treat or prevent T2DM. © 2018 Society of Chemical Industry.

Antihyperalgesic Effects of Indomethacin, Ketorolac, and Metamizole in Rats: Effects of Metformin.

Preclinical Research Metformin-dependent mechanisms have been implicated in the antinociceptive effect of some non-steroidal anti-inflammatory drugs (NSAIDs). In this study, the effect of local peripheral or systemic administration of metformin on the local peripheral or systemic antinociception induced by indomethacin, ketorolac and metamizole was assessed in the rat carrageenan-induced thermal hyperalgesia model. Rats were injected with carrageenan (1%, 50 µl) into the right hindpaw which reduced paw withdrawal latency, a measure of thermal hyperalgesia. Local peripheral or systemic administration of indomethacin, ketorolac or metamizole dose-dependently reduced carrageenan-induced thermal hyperalgesia. Local peripheral pre-treatment with metformin (800 µg/paw) partially inhibited the anti-hyperalgesic effect of indomethacin (200 µg/paw) and metamizole (200 µg/paw), but not that of ketorolac (200 µg/paw). In contrast, systemic pre-treatment with metformin (200 mg/kg) attenuated the antihyperalgesic effect of metamizole (10 mg/kg), but not that observed with either indomethacin (10 mg/kg) or ketorolac (10 mg/kg). These findings suggest that some but not all NSAIDs have effects mediated by metformin-dependent mechanisms. Drug Dev Res 78 : 98-104, 2017. ©2017 Wiley Periodicals, Inc.

Acute Hypoglycemic and Antidiabetic Effect of Teuhetenone A Isolated from Turnera diffusa.

Diabetes mellitus is a chronic degenerative disease that causes long-term complications and represents a serious public health problem. Turnera diffusa (damiana) is a shrub that grows throughout Mexico and is traditionally used for many illnesses including diabetes. Although a large number of plant metabolites are known, there are no reports indicating which of these are responsible for this activity, and this identification was the objective of the present work. Through bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa, teuhetenone A was isolated and identified as the main metabolite responsible for the plant's hypoglycemic activity. Alpha-glucosidase inhibitory activity and cytotoxicity of this metabolite were determined. Hypoglycemic and antidiabetic activities were evaluated in a murine model of diabetes in vivo, by monitoring glucose levels for six hours and comparing them with levels after administering various controls. Teuhetenone A was not cytotoxic at the tested concentrations, and did not show inhibitory activity in the glucosidase test, and the in vivo assays showed a gradual reduction in glucose levels in normoglycemic and diabetic mice. Considering these results, we suggest that teuhetenone A has potential as an antidiabetic compound, which could be further submitted to preclinical assays.

Fosinopril Prevents the Development of Tactile Allodynia in a Streptozotocin-Induced Diabetic Rat Model.

The aim of this study was to evaluate fosinopril-induced changes in hemodynamic parameters and tactile allodynia in a rat model of diabetes. Diabetes was induced by streptozotocin (STZ; 50 mg/kg, i.p.) in male Wistar rats. STZ produced hyperglycemia, weight loss, polydipsia, polyphagia, and polyuria as well as long-term arterial hypotension, bradycardia, and tactile allodynia at 10-12 weeks. Daily administration of the angiotensin converting enzyme inhibitor, fosinopril (25 mg/kg, p.o., for 11 weeks) partially reduced the loss of body weight, decreased hyperglycemia, and systolic blood pressure in diabetic rats. Likewise, systemic administration of fosinopril prevented the development and maintenance of tactile allodynia in STZ-induced diabetic rats. These data suggest that fosinopril may have a role in the pharmacotherapy of diabetic neuropathic pain.

Early, middle, or late administration of zoledronate alleviates spontaneous nociceptive behavior and restores functional outcomes in a mouse model of CFA-induced arthritis.

This study was performed to evaluate whether early, middle, or late treatment of zoledronate, an approved bisphosphonate that blocks bone resorption, can reduce nociceptive behaviors in a mouse arthritis model. Arthritis was produced by repeated intra-articular knee injections of complete Freund's adjuvant (CFA). A dose-response curve with zoledronate (3, 30, 100, and 300 µg/kg, i.p., day 4 to day 25, twice weekly for 3 weeks) was performed, and the most effective dose of zoledronate (100 µg/kg, i.p.) was initially administered at different times of disease progression: day 4 (early), day 15 (middle), or day 21 (late) and continued until day 25 after the first CFA injection. Flinching of the injected extremity (spontaneous nociceptive behavior), vertical rearings and horizontal activity (functional outcomes), and knee edema were assessed. Zoledronate improved both functional outcomes and reduced flinching behavior. At day 25, the effect of zoledronate on flinching behavior and vertical rearings was greater in magnitude when it was given early or middle rather than late in the treatment regimen. Chronic zoledronate did not reduce knee edema in CFA-injected mice nor functional outcomes in naïve mice by itself. These results suggest that zoledronate may have a positive effect on arthritis-induced nociception and functional disabilities.

Nociplastic pain mechanisms and toll-like receptors as promising targets for its management.

ABSTRACT: Nociplastic pain, characterized by abnormal pain processing without an identifiable organic cause, affects a significant portion of the global population. Unfortunately, current pharmacological treatments for this condition often prove ineffective, prompting the need to explore new potential targets for inducing analgesic effects in patients with nociplastic pain. In this context, toll-like receptors (TLRs), known for their role in the immune response to infections, represent promising opportunities for pharmacological intervention because they play a relevant role in both the development and maintenance of pain. Although TLRs have been extensively studied in neuropathic and inflammatory pain, their specific contributions to nociplastic pain remain less clear, demanding further investigation. This review consolidates current evidence on the connection between TLRs and nociplastic pain, with a specific focus on prevalent conditions like fibromyalgia, stress-induced pain, sleep deprivation-related pain, and irritable bowel syndrome. In addition, we explore the association between nociplastic pain and psychiatric comorbidities, proposing that modulating TLRs can potentially alleviate both pain syndromes and related psychiatric disorders. Finally, we discuss the potential sex differences in TLR signaling, considering the higher prevalence of nociplastic pain among women. Altogether, this review aims to shed light on nociplastic pain, its underlying mechanisms, and its intriguing relationship with TLR signaling pathways, ultimately framing the potential therapeutic role of TLRs in addressing this challenging condition.

Neuroplasticity of sensory and sympathetic nerve fibers in a mouse model of a painful arthritic joint.

OBJECTIVE: Many forms of arthritis are accompanied by significant chronic joint pain. This study was undertaken to investigate whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathologic reorganization. METHODS: A painful arthritic knee joint was produced by injection of Freund's complete adjuvant (CFA) into the knee joint of young adult mice. CFA-injected mice were then treated systemically with vehicle or anti-NGF antibody. Pain behaviors were assessed, and at 28 days following the initial CFA injection, the knee joints were processed for immunohistochemistry analysis using antibodies against calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kd (NF200; sensory nerve fibers), growth-associated protein 43 (GAP-43; sprouted nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), CD31 (endothelial cells), or CD68 (monocyte/macrophages). RESULTS: In CFA-injected mice, there was a significant increase in the density of CD68+ macrophages, CD31+ blood vessels, and CGRP+, NF200+, GAP-43+, and TH+ nerve fibers in the synovium, as well as a significant increase in joint pain-related behaviors. None of these findings were observed in sham-injected mice. Administration of anti-NGF reduced these pain-related behaviors and the ectopic sprouting of nerve fibers, but had no significant effect on the increase in density of CD31+ blood vessels or CD68+ macrophages. CONCLUSION: These findings demonstrate that ectopic sprouting of sensory and sympathetic nerve fibers occurs in the painful arthritic joint and may be involved in the generation and maintenance of arthritic pain.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents.

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

Characterization of pain-related behaviors, changes in bone microarchitecture and sensory innervation induced by chronic cadmium exposure in adult mice.

Because of the relative lack of understanding of the neurobiological mechanisms that drive toxic effects of cadmium in bone, the purpose of this study was to characterize a preclinical model of chronic cadmium exposure. Adult male C57BL/6 J mice were exposed to cadmium 25 mg/L (as CdCl2) in drinking water for 16 weeks. During this time, pain-related behaviors including hindpaw mechanical sensitivity and vertical rears were evaluated every four weeks. We assessed changes in bone microarchitecture at the femoral neck and L5 vertebra by microcomputed tomography and quantified the density of nerve fibers expressing PGP 9.5 (a pan-neuronal marker) and CGRP (a marker of sensory nerve fibers subfamily) at the femoral neck and glabrous skin of the hindpaw using immunohistochemistry. Cadmium exposure produced mechanical hypersensitivity in both hindpaws along with decreased rearing activity (surrogate for musculoskeletal-related pain) without affecting the horizontal activity (a measure of locomotor behavior) in comparison to the control group. Intraperitoneal acute treatment with morphine and gabapentin reversed pain-related behaviors in cadmium-exposed mice. Furthermore, exposure to cadmium resulted in significant trabecular bone deterioration at the femoral neck and L5 vertebra. We also observed a significant reduction in the density of both CGRP+ and PGP 9.5+ nerve fibers in the femoral neck, but not in the hindpaw glabrous skin, suggesting tissue-dependent neurotoxicity. This model may help in developing a mechanism-based understanding of the factors that generate and maintain musculoskeletal pain and bone loss caused by chronic cadmium exposure and in translating these findings into new therapies for treating cadmium-induced bone toxicity.

Vertical transmission of Trypanosoma cruzi in the Province of Choapa, IV Region, Chile: Preliminary Report (2005-2008).

Congenital Chagas disease acquired special importance in Chile after the certification of the control of Triatoma infestans and transmission by blood donors affected with Trypanosoma cruzi. In order to establish adequate protocols for intervention and control in infected mother-neonate pairs in endemic zones of Chagas disease, we present partial results (2005-2008) of a pilot project which is being carried out in the Province of Choapa, IV Region, Chile, whose objectives are: determine the current prevalence of the disease in pregnant women, estimate the incidence of vertical transmission of T. cruzi to newborns, determine the lineages of the parasite present in mothers who do and do not transmit the disease, determine the prevalence of Chagas disease in maternal grandmothers of neonates and study placental histopathology. Preliminary results indicated that in this study period, 3.7% of the women who gave birth in the Province have Chagas disease and 2.5% of their newborns were infected. The most frequent T. cruzi genotypes found in mothers studied during pregnancy were TCI and TCIId, either alone or in mixed infections. A high percentage (74.3%) of the grandmothers studied was infected with the parasite. In 29 placentas from mothers with Chagas disease we observed edema, necrosis, fibrinoid deposits and slight lymphoplasmocyte infiltration. In three placentas we found erythroblastosis and in one of them amastigote forms of T. cruzi; this was one of the cases of congenital infection. The evaluation of the diagnostic and control protocols generated will allow us to determine if it has been possible to modify the natural history of vertical transmission of T. cruzi in Chile.

Characterization of Mechanical Allodynia and Skin Innervation in a Mouse Model of Type-2 Diabetes Induced by Cafeteria-Style Diet and Low-Doses of Streptozotocin.

Background: Painful distal symmetrical polyneuropathy (DPN) is a frequent complication of type-2 diabetes mellitus (T2DM) that commonly presents as neuropathic pain and loss of skin nerve fibers. However, there are limited therapies to effectively treat DPN and many of the current animal models of T2DM-induced DPN do not appear to mirror the human disease. Thus, we validated a DPN mouse model induced by a cafeteria-style diet plus low-doses of streptozotocin (STZ). Methods: Female C57BL/6J mice were fed either standard (STD) diet or obesogenic cafeteria (CAF) diet for 32 weeks, starting at 8 weeks old. Eight weeks after starting diets, CAF or STD mice received either four low-doses of STZ or vehicle. Changes in body weight, blood glucose and insulin levels, as well as oral glucose- and insulin-tolerance tests (OGTT and ITT) were determined. The development of mechanical hypersensitivity of the hindpaws was determined using von Frey filaments. Moreover, the effect of the most common neuropathic pain drugs was evaluated on T2DM-induced mechanical allodynia. Finally, the density of PGP -9.5+ (a pan-neuronal marker) axons in the epidermis from the hindpaw glabrous skin was quantified. Results: At 22-24 weeks after STZ injections, CAF + STZ mice had significantly higher glucose and insulin levels compared to CAF + VEH, STD + STZ, and STD + VEH mice, and developed glucose tolerance and insulin resistance. Skin mechanical sensitivity was detected as early as 12 weeks post-STZ injections and it was significantly attenuated by intraperitoneal acute treatment with amitriptyline, gabapentin, tramadol, duloxetine, or carbamazepine but not by diclofenac. The density of PGP-9.5+ nerve fibers was reduced in CAF + STZ mice compared to other groups. Conclusion: This reverse translational study provides a painful DPN mouse model which may help in developing a better understanding of the factors that generate and maintain neuropathic pain and denervation of skin under T2DM and to identify mechanism-based new treatments.

Acute toxicity and mutagenic activity of Mexican plants used in traditional medicine.

The present work was undertaken to determine safety parameters of selected Mexican medicinal plants chosen on the basis of their frequency of medicinal use and commercial importance. The medicinal herbs included Amphipteryngium adstringens, Hintonia standleyana, Hintonia latiflora, Piper sanctum, Haemathoxylon brasiletto, Iostephane heterophylla, Valeriana procera, Arracacia tolucensis, Brickellia veronicaefolia, Scaphyglottis livida, Exostema caribaeum, Hippocratea excelsa, Ligusticum porteri, Poliomintha longiflora and Gnaphalium sp. In the acute toxicity studies in mice performed according to the Lorke procedure, Exostema caribaeum, Hippocratea excelsa, Ligusticum porteri and Poliomintha longiflora were the most toxic with LD(50) values between 1085 and 2mg/kg. The Ames test revealed that Gnaphalium sp. and Valeriana procera extracts induced mutations of S. typhimurium TA98 with or without the S9 microsomal fraction, and TA100 in the presence of the enzymatic fraction, respectively. The tincture of Valeriana procera, however, was non-mutagenic. Finally, in the Artemia salina lethality test Brickellia veronicaefolia, Arracacia tolucensis, Poliomintha longiflora and Piper sanctum caused significant mortality of the crustacean larvae with LC(50) in the range of 37-227 microg/mL.

Reorganization of extracellular matrix in placentas from women with asymptomatic chagas disease: mechanism of parasite invasion or local placental defense?

Chagas disease, produced by the protozoan Trypanosoma cruzi (T. cruzi), is one of the most frequent endemic diseases in Latin America. In spite the fact that in the past few years T. cruzi congenital transmission has become of epidemiological importance, studies about this mechanism of infection are scarce. In order to explore some morphological aspects of this infection in the placenta, we analyzed placentas from T. cruzi-infected mothers by immunohistochemical and histochemical methods. Infection in mothers, newborns, and placentas was confirmed by PCR and by immunofluorescence in the placenta. T. cruzi-infected placentas present destruction of the syncytiotrophoblast and villous stroma, selective disorganization of the basal lamina, and disorganization of collagen I in villous stroma. Our results suggest that the parasite induces reorganization of this tissue component and in this way may regulate both inflammatory and immune responses in the host. Changes in the ECM of placental tissues, together with the immunological status of mother and fetus, and parasite load may determine the probability of congenital transmission of T. cruzi.

Blockade of peripheral and spinal Na+/H+ exchanger increases formalin-induced long-lasting mechanical allodynia and hyperalgesia in rats.

The Na(+)/H(+) exchanger (NHE) is involved in the regulation of intracellular pH and volume by mediating the electroneutral transport of H(+) against an influx of Na(+) ions. Since NHE1 regulates pH in neurons and astrocytes and it is expressed in nociceptive nerve fibers, it is likely that NHE may modulate neuronal excitability and pain transmission. The purpose of this study was to assess the participation of peripheral and spinal NHE in the secondary allodynia/hyperalgesia induced by formalin. In addition, we determined whether formalin injection modifies the expression of NHE1 in lumbar dorsal root ganglia (DRG) and dorsal spinal cord. Subcutaneous injection of 0.5% formalin into the dorsal surface of the hind paw produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-lasting bilateral secondary mechanical allodynia/hyperalgesia. Peripheral and intrathecal pre-treatment (-10min) with selective NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3-30µM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3-30µM) and [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine dihydrochloride (zoniporide, 0.03-3µM) significantly increased 0.5% formalin-induced bilateral long-lasting secondary allodynia/hyperalgesia. Contrariwise, local peripheral or intrathecal post-treatment (day 6 postinjection) with these NHE inhibitors did not affect formalin-induced nociceptive behaviors. Formalin injection reduced NHE1 expression in ipsilateral and contralateral spinal dorsal horns from day 1 to 12. In addition, formalin diminished NHE1 protein expression in DRG at day 12. These results suggest that NHE1 plays a role in pain processing at peripheral and spinal levels in formalin-induced long-lasting nociceptive behaviors. Additionally, these results suggest that proteins involved in pH regulation could be targets for the development of new analgesic drugs.

Role of the spinal Na+/H+ exchanger in formalin-induced nociception.

This study assessed the role of the Na(+)/H(+) exchanger (NHE) in the formalin-induced nociception as well as the expression of the NHE isoform 1 (NHE1) in the rat spinal cord by using immunohistochemistry. Rats received a 50µl injection of diluted formalin (0.5%). Nociceptive behavior was quantified as the number of flinches of the injected paw. Intrathecal administration of the partially selective NHE1 inhibitors DMA, EIPA (0.3-30µM/rat) and the selective NHE1 inhibitor zoniporide (0.03-3µM/rat) significantly increased formalin-induced flinching behavior in a dose-dependent manner during both phases of the test. Immunohistochemical analysis of the rat lumbar spinal cord showed that NHE1 was mainly expressed in the lamina I of the dorsal horn of the spinal cord. Double immunofluorescence staining showed co-localization of NHE1 with the peptide-rich sensory nerve fiber markers, substance P and calcitonin gene-related peptide, but not with markers of neuronal cell bodies (NeuN), microglia (OX-42) or astroglia (GFAP). Collectively, these pharmacological and anatomical results suggest that spinal NHE1 plays a role in formalin-induced nociception acting as a protective protein extruding H(+).

Role of peripheral 5-HT(4), 5-HT(6), and 5-HT(7) receptors in development and maintenance of secondary mechanical allodynia and hyperalgesia.

The role of 5-hydroxytryptamine (5-HT)(4), 5-HT(6), and 5-HT(7) receptors in formalin-induced secondary allodynia and hyperalgesia in rats was assessed. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia. Pretreatment (-10min) with cromoglycate (195-1950nmol/paw) partially inhibited acute nociceptive behaviors and completely prevented secondary allodynia and hyperalgesia on day 6 after injection. Ipsilateral peripheral pretreatment with the selective 5-HT(4) (ML-10302, 1-100nmol/paw), 5-HT(6) (EMD-386088, 0.001-0.01nmol/paw), and 5-HT(7) (LP-12, 0.01-100nmol/paw) receptor agonists significantly increased secondary allodynia and hyperalgesia in both paws. In contrast, ipsilateral peripheral pretreatment with the selective 5-HT(4) (GR-125487, 1-100nmol/paw), 5-HT(6) (SB-258585, 0.00001-0.001nmol/paw), and 5-HT(7) (SB-269970, 0.1-10nmol/paw) receptor antagonists significantly prevented formalin-induced secondary allodynia and hyperalgesia in both paws. The pronociceptive effect of ML-10302 (100nmol/paw), EMD-386088 (0.01nmol/paw), and LP-12 (100nmol/paw) were completely prevented by GR-125487 (5-HT(4) antagonist, 1nmol/paw), SB-258585 (5-HT(6) antagonist, 0.00001nmol/paw), and SB-269970 (5-HT(7), antagonist, 0.01nmol/paw), respectively. Ipsilateral peripheral posttreatment with cromoglycate or GR-125487 (1-100nmol/paw), SB-258585 (0.001-0.1nmol/paw), and SB-269970 (0.1-10nmol/paw) reversed formalin-induced secondary allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT(4), 5-HT(6), and 5-HT(7) receptors participate in the development and maintenance of formalin-induced long-term secondary allodynia and hyperalgesia in the rat. 5-hydroxytryptamine (5-HT) released in peripheral tissues after formalin injection sensitized primary afferent neurons via 5-HT(4), 5-HT(6), and 5-HT(7) receptors, leading to development and maintenance of secondary allodynia and hyperalgesia.

Preventive or late administration of anti-NGF therapy attenuates tumor-induced nerve sprouting, neuroma formation, and cancer pain.

Early, preemptive blockade of nerve growth factor (NGF)/tropomyosin receptor kinase A (TrkA) attenuates tumor-induced nerve sprouting and bone cancer pain. A critical unanswered question is whether late blockade of NGF/TrkA can attenuate cancer pain once NGF-induced nerve sprouting and neuroma formation has occurred. By means of a mouse model of prostate cancer-induced bone pain, anti-NGF was either administered preemptively at day 14 after tumor injection when nerve sprouting had yet to occur, or late at day 35, when extensive nerve sprouting had occurred. Animals were humanely killed at day 70 when, in vehicle-treated animals, significant nerve sprouting and neuroma formation was present in the tumor-bearing bone. Although preemptive and sustained administration (days 14-70) of anti-NGF more rapidly attenuated bone cancer nociceptive behaviors than late and sustained administration (days 35-70), by day 70 after tumor injection, both preemptive and late administration of anti-NGF significantly reduced nociceptive behaviors, sensory and sympathetic nerve sprouting, and neuroma formation. In this model, as in most cancers, the individual cancer cell colonies have a limited half-life because they are constantly proliferating, metastasizing, and undergoing necrosis as the parent cancer cell colony outgrows its blood supply. Similarly, the sensory and sympathetic nerve fibers that innervate the tumor undergo sprouting at the viable/leading edge of the parent tumor, degenerate as the parent cancer cell colony becomes necrotic, and resprout in the viable, newly formed daughter cell colonies. These results suggest that preemptive or late-stage blockade of NGF/TrkA can attenuate nerve sprouting and cancer pain.