What Effect Will The Saline Injections Have On The Control Rat's Vertebral Bone Density

Neurosci Lett. Author manuscript; available in PMC 2012 May 9.

Published in final edited form as:

PMCID: PMC3119508

NIHMSID: NIHMS284112

Hypersensitivity and Hyperinnervation of the Rat Hind Manus Following Carrageenan-induced Inflammation

Anuradha Chakrabarty

¹ Department of Molecular and Integrative Physiology, University of Kansas Medical Centre, Kansas City, KS 66160

³ Kansas Intellectual and Developmental Disabilities Research Middle, University of Kansas Medical Center, Kansas City, KS 66160

^iv Institute for Neurological Disorders, University of Kansas Medical Center, Kansas City, KS 66160

Kenneth E. McCarson

^two Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Eye, Kansas City, KS 66160

^three Kansas Intellectual and Developmental Disabilities Research Eye, University of Kansas Medical Center, Kansas Urban center, KS 66160

^iv Found for Neurological Disorders, University of Kansas Medical Center, Kansas City, KS 66160

Peter G. Smith

¹ Department of Molecular and Integrative Physiology, Academy of Kansas Medical Centre, Kansas City, KS 66160

³ Kansas Intellectual and Developmental Disabilities Enquiry Eye, University of Kansas Medical Eye, Kansas Urban center, KS 66160

^iv Plant for Neurological Disorders, University of Kansas Medical Center, Kansas Metropolis, KS 66160

Abstract

Studies of human tissue show that many chronic hurting syndromes are accompanied by abnormal increases in numbers of peripheral sensory nerve fibers. It is not known if sensory nerve sprouting occurs as a result of inflammation nowadays in these conditions, or other factors such every bit infection or extensive tissue impairment. In the nowadays study, we used a well established model of inflammation to examine cutaneous innervation density in relation to mechanical and thermal hypersensitivity. Developed female person rats were ovariectomized to eliminate fluctuations in female reproductive hormones and one week later, a hind hand was injected with carrageenan or saline vehicle. Behavioral testing showed that saline vehicle injection did not alter thermal or mechanical thresholds compared to pre-injection baselines. Carrageenan injections resulted in markedly reduced paw withdrawal thresholds at 24 and 72 h after injection; this was accompanied past increased mechanical sensitivity of the contralateral paw at 72h. Assay of innervation density using PGP9.5 as a pan-neuronal marker at 72h showed that inflammation resulted in a ii-fold increment in cutaneous innervation density. We conclude that inflammation lonely is sufficient to induce sprouting of sensory cutaneous axon endings leading local tissue hyperinnervation, which may contribute to hypersensitivity that occurs in painful inflammatory atmospheric condition.

Keywords: Pain, sensory nerves, axon sprouting, skin

Introduction

Mechanisms of chronic pain are poorly understood, and several factors have been identified that may contribute to enhanced peripheral nociceptor sensitivity. Persistent pain is postulated to involve alterations of neuronal ion channels [4, eleven] and membrane receptors [41], and may involve recruitment of otherwise dormant nociceptors [23]. There also is bear witness that sensitization of both peripheral [39] and central neurons [38, 40] may contribute to hyperalgesia [33] and allodynia [24]. Therefore, multiple factors are thus far implicated in the establishment of chronic hurting.

Accumulating evidence in humans suggests that structural remodeling of peripheral sensory fibers may be an additional gene in some types of hurting. For example, peripheral neuropathic pain is often accompanied by decreased cutaneous axon density, leading to centrally mediated pain [39]. Conversely, an abnormal increase in numbers of nerve fibers (hyperinnervation) is reported to occur in several types of musculo-skeletal pain including Achilles tendinitis [2, 30], chronic knee hurting [28, 29] and degenerative disk pain [8, 12]. Other pain syndromes associated with hyperinnervation include chronic appendicitis [ten], deep infiltrating endometriosis [35], mastodynia [14], and vulvodynia [5, half dozen, 34]. Therefore, sensory hyperinnervation appears to be a mutual characteristic associated with a number of chronically painful atmospheric condition.

While the mechanisms leading to hyperinnervation are unclear, inflammation is a mutual thread in all disease syndromes in which hyperinnervation is reported to occur. Thus, inflammation may be an initiating factor leading to sensory axon proliferation. The inflammatory milieu contains cytokines, chemokines, and growth factor proteins [22] which, alone or in combination, may promote sensory axon sprouting. Nevertheless, it is every bit yet unclear whether hyperinnervation occurs equally a upshot of inflammation or is secondary to extensive tissue injury and repair, infection, or degenerative processes that also occur in these human illness weather. In the nowadays report nosotros assessed whether hyperinnervation occurs in a well-established model of inflammation and accompanies behavioral hypersensitivity.

Materials and methods

All beast protocols and procedures were in accord with the NIH guidelines for the care and use of laboratory animals and were approved past the Kansas Academy Medical Heart Beast Care and Use Committee. Twelve female person Sprague-Dawley rats (Harlan Teklad, Madison, WI) at approximately 60 d and weighing 190 – 200 g were anesthetized (i.p. injection of 70 mg/kg ketamine HCl [Ketaject] and half dozen mg/kg xylazine [Xyla-Ject]) and bilaterally ovariectomized under hygienic conditions; ovariectomy eliminates cyclic fluctuations in serum reproductive hormone levels that are known to influence behavioral sensitivity [ix, 27]. Later 7 days, rats were randomly distributed to 2 groups of half dozen each; one received a subcutaneous plantar injection into the left manus of 0.1 ml of 2% (westward/v) λ-carrageenan (Sigma-Aldrich, St. Louis, MO) in 0.9% sterile saline, while rats in the second group received identical injections of sterile isotonic saline vehicle. All injections were fabricated by inserting a 29 gauge needle into the posterior region of the plantar surface and delivering the injectate into the approximate center of the plantar surface.

Behavioral sensitivity was assessed by first measuring mechanical withdrawal threshold (MWT) using an electronic von Frey anesthesiometer (Model 2390; IITC Inc., Woodland Hills, CA) so thermal withdrawal latency (TWL) using a Paw Thermal Stimulator Instrument (University of California, San Diego). All behavioral tests were performed between viii:xxx – 11:30 AM. For MWT, rats were acclimated for 30 min inside a Plexiglas box on a steel mesh floor and analyses performed using an electronic von Frey apparatus. Stimulation was applied to the center of the hind paw in an upward move with the von Frey filament until foot withdrawal occurred, and withdrawal threshold was automatically recorded. The procedure was repeated three times at 3–5 min intervals for each hind paw and the average calculated. Rats were and so acclimated for xxx min in individual Plexiglas boxes on a drinking glass plate maintained at 30°C. The fourth dimension to withdraw the hind manus from a high-intensity light beam was recorded automatically. The test was performed three times at three–five min intervals on each mitt, and the average value calculated for each session. Behavioral tests were performed at 24h prior to (n=6) and 24 and 72h post-carrageenan or vehicle injection (n=iii each). Following behavioral testing at 72h, rats were deeply anesthetized with pentobarbital (150mg/kg i.p.), and plantar surface skin from both hind paws removed and fixed in Zamboni's fixative at 4°C overnight, rinsed in phosphate buffered saline (0.01M, pH 7.iv) for several days, and cryoprotected overnight in 20% sucrose. Tissues were mounted in Tissue Tek OCT chemical compound, snap-frozen and cryosectioned at xx μm perpendicular to the long axis of the hand. Sections collected from the center of the region where the injectate was deposited (four mm proximal to the junction with the toes) were blocked with ten% normal caprine animal serum (Jackson Immuno Research Laboratory, West Grove, PA), incubated overnight with rabbit IgG directed against the pan-neuronal mark PGP9.5 (ane:400; Serotec, Raleigh, NC), rinsed with PBS containing 0.3% triton Ten-100, and incubated with Cy2 conjugated goat anti-rabbit (Jackson) at room temperature for ane h. Negative controls included primary antisera preabsorbtion to blocking peptides, oestrus inactivation and antibody omission.

Average innervation density was measured in three sample fields per section; 1 sample field (149,188 μm²) was from the section's eye, directly over the site of injectate deposition, and i each from the lateral parts of the section respective to the margins of the inflamed tissue. For each subject, analyses were obtained from 3 sections taken at ane mm intervals, with 6 subjects each analyzed in the saline- and carageenan- injected groups. Images were captured with a Nikon Eclipse 80i microscope using a Nikon Fluor 20X/0.l DIC Yard/N2 objective, and a Nikon DSFi1 camera. A stereological filigree (AnalySis v.iii.2) with intersects at x μm intervals was superimposed over each image. The number of intersects overlying the epidermis was counted, and the number of points overlying PGP-immunoreactive (-ir) axons was besides adamant in each field. Credible percentage of epidermal area occupied past PGP-ir axon was calculated by dividing intersects overlying axons by intersects overlying the tissue within the sample field. To obtain the apparent surface area occupied by axons, the fraction of epidermal tissue occupied by PGP-ir fretfulness was multiplied by the total expanse of the epidermal tissue compartment within each sample surface area. Values were normalized to the length of epidermis sampled inside each field and expressed every bit PGP9.5-ir axon area (μmⁱⁱ) per mm. Values from the 9 sample regions for each paw were averaged. Information obtained from behavioral and stereological analyses are presented equally mean ± SEM. Statistical comparisons were made using Educatee's t-test or one way ANOVA (all data were normally distributed). Mail-hoc comparisons were made using the Student-Newman-Keuls test and differences considered significant at p ≤ 0.05.

Results

Intraplantar saline injections resulted in transient mild vasodilation before long after injection, with no obvious inflammation at 24h or 72h. Carrageenan injection resulted in marked vasodilation followed by significant edema and hand licking though 24h, resolving slightly by 72h postal service-injection. The contralateral paws appeared normal and unaffected.

At 24 h prior to treatment, TWL was 10.0±0.7 sec for the left paw (Fig. i) and 10.5±0.four sec for the right paw (not shown). TWL of the saline-injected left paws were comparable to the uninjected control paws, and were unchanged through 72h post-saline injection (Fig. 1).

An external file that holds a picture, illustration, etc. Object name is nihms284112f1.jpg

Thermal sensitivity was assessed past measuring time required for rats to withdraw their paws in response to warming (Thermal Withdrawal Latency). Baseline measurements were made from the left paw of vi rats 24 h prior to injections (No Treatment, −24). Measurements from the left hind hand 24 hours later on saline (Vehicle, northward=3) or carrageenan (Carr, n=3) injection showed increased sensitivity. At 72 h later injection, the paw ipsilateral (Ipsi) to carrageenan injection showed sustained hypersensitivity relative to ipsilateral vehicle injection and baseline measurements. Responses from paws contralateral to injection (Contra) were comparable. Carrageenan vs. Vehicle at 24–72 h, p<0.001 by ANOVA and Student-Newman-Keuls test.

Carrageenan injection markedly decreased TWL relative to uninjected and saline injected paws. TWL post-obit carrageenan injection was reduced by 58% relative to saline injection after 24h, and remained decreased for 72h (Fig. 1, p<0.001 at 24h and 72h). No differences were observed in TWL measured at 72 h from hind paws contralateral to carrageenan injection.

Mechanical withdrawal thresholds measured from the left hind paw prior to injection were 28.0±ii.1gm (Fig. 2), which was comparable to the contralateral paw (not shown). In vehicle-injected rats, no significant differences were observed for MWT at 24h and 72h mail service-injection. Carrageenan injection resulted in a significant subtract in MWT relative to saline injection at 24h (6.eight±0.viii vs.26.i±0.9 gm; p<0.001) and 72h (viii.2 ±1.vi vs 23.8±1.7 gm; p<0.001; Fig. two). MWTs from hind paws contralateral to carrageenan injection were lower than those contralateral to vehicle injection at 72h (p = 0.009, Fig. two).

An external file that holds a picture, illustration, etc. Object name is nihms284112f2.jpg

Mechanical sensitivity was assessed past electronic von Frey anesthesiometry to determine force required to elicit a withdrawal response. Baseline measurements were obtained from the manus 24 h prior to injections (No Treatment, −24, due north=6). Measurements from the left hind mitt 24 hours after injection showed heightened sensitivity following carrageenan (Carr, n=3) relative to saline (Vehicle, n=three) injection (p<0.001). At 72 h subsequently injection, the hand ipsilateral (Ipsi) to carrageenan injection showed continued hypersensitivity relative to ipsilateral vehicle injection and baseline measurements (n=3/group, p<0.001). The paw contralateral (Contra) to the carrageenan injection showed increased sensitivity relative to the paw contralateral to saline injection at 72 h (p = 0.009).

Sections of the plantar skin from uninjected right paws immunostained for PGP9.5 showed nerve cobweb bundles oriented longitudinally along the epidermal-dermal interface, with fine axonal branches extending perpendicularly for variable distances into the epidermis; smaller numbers of fiber bundles were present within the dermal region most the epidermis (Fig. 3A). The density of innervation, as adamant past sectional axon surface area per unit length of skin, did non differ betwixt contralateral paws of rats injected with either saline or carrageenan (Fig. 3A, D, E). Saline injection (Fig. 3B) did non significantly affect total innervation density of the epidermis compared to the uninjected paw skin (Fig. 3E). In dissimilarity, carrageenan injection (Fig. 3C) resulted in a two-fold increase in innervation relative to the contralateral uninjected hand (Fig. 3D, East; p<0.001) and approximately a 70% increase (p = 0.022) over that of saline injection (Fig. 3B, Due east).

An external file that holds a picture, illustration, etc. Object name is nihms284112f3.jpg

Confocal micrographs of PGP9.5-immunoreactive axons in footpad epidermis 72 h following saline or carrageenan injection. A) Uninjected footpad epidermis contralateral saline injection. B) Saline injected footpad epidermis. C) Carrageenan injected footpad epidermis. D) Uninjected footpad epidermis contralateral to carrageenan injection. Calibration bar in D = 40 μm. Due east) Quantitative analysis of footpad epidermis revealed that axon density was increased post-obit carrageenan injection relative to the contralateral foot or saline injection (p <0.001, n=6 per grouping).

Discussion

Carrageenan injection into the rat plantar hind paw is a well established model of inflammation. Carrageenans are linear sulfated polysaccharides derived from blood-red seaweeds, which possess potent inflammatory properties. Carrageenan injections into the plantar aspect of the rodent paw were first used by Winter et al. every bit an edema-based assay for evaluating anti-inflammatory drugs [37]. Afterward, this model has been used widely to evoke thermal hyperalgesia and mechanical allodynia in rodents [17, 25, 31]. Following carrageenan injection, edema and behavioral hypersenstitivity are established by four h after injection and last through at least 96 h [ane, 20, 25].

To confirm that carageenan injections were accompanied past increased sensitivity in this study, behavioral testing was performed 24 h before and 24 h and 72 h subsequently injections. A difference betwixt this study and others is the utilize of ovariectomized rats. Female reproductive hormones tin modulate and alter carrageenan-induced sensitivity [32], which tin can present a confounding variable in intact cycling animals. In the nowadays study we circumvented this effect by using female person rats who received short-term ovariectomies, a procedure that essentially eliminates variations in ovarian hormone levels. Nosotros plant that carageenan injection produces substantial increases in both thermal and mechanical sensitivity at 24 and 72h, a finding consistent with earlier reports in intact rats [17, 31]. This confirms that relatively stable and persistent thermal and mechanical hypersensitivity is elicited in this model.

A central question nosotros wished to address was whether inflammation alone is sufficient to induce hyperinnervation. Hyperinnervation accompanies many painful inflammatory weather, equally revealed in tissue biopsies and in cadaveric specimens. However inflammation in these conditions generally does not occur in isolation but rather as a secondary characteristic induced by extensive tissue harm, rejection, or main bacterial or viral infections. In the present study, we show that within 72h of inflammatory challenge, the epithelial tissue compartment shows marked increases in numbers of nervus fibers. It is noteworthy that our neuronal marker, PGP9.5, labels all intact axons and its expression levels typically do not change in weather short of degeneration [36]. Immunostained axons are likely to reflect both the abundant non-peptidergic and less arable peptidergic sensory fibers that populate the rodent epidermis [26]. The finding that numbers of cutaneous sensory axons are increased by injection of carrageenan just not saline argues that axon proliferation occurs as a upshot of the inflammatory process rather than simply physical trauma. Thus, inflammation in the absenteeism of typically related factors (e.one thousand., injury, infection) appears to be sufficient to induce cutaneous hyperinnervation.

It is unclear whether this increased sensory axon density contributes to the increased mechanical and thermal sensitivity we observed, only several lines of testify propose that it may. There is evidence that actively growing axons prove greater excitability than do quiescent axons [fifteen, 18]. Similarly, neurons with more circuitous geometries, every bit occurs following axon sprouting, too show greater excitability [xix]. In the case of neurons projecting to the site of inflammation, axons are exposed to a diverseness of excitatory molecules including prostaglandin E2 [16], adenosine [21] and NGF [3], and greater axonal branching is likely to upshot in greater summation of depolarizing potentials evoked by these local factors. Branching is also likely to give ascent to greater numbers of locally activated neuropeptide-releasing sites, farther contributing to the inflammatory procedure. Accordingly, it is quite possible that hyperinnervation acts in concert with other factors, such every bit changes in sodium aqueduct expression and excitability [4], to increase pain sensitivity under conditions of inflammation. Indeed, the increased mechanical sensitivity seen in the paw contralateral to the carrageenan injection is consistent with sensitization occurring centrally or equally a outcome of systemic peripheral factors.

Findings of the present study support the idea that sensory axons respond to inflammation by sprouting new branches to increase tissue innervation density. As noted, this is now believed to be a feature mutual to many pain syndromes in humans. Information technology remains unclear as to the extent to which hyperinnervation may contribute to sustaining chronic pain after acute inflammation subsides. All the same, in cases of provoked vulvodynia (vulvar vestibulitis) characterized by proliferation of vestibular sensory axons [5, 6, 34], excision of the tissue containing the abundant fibers provides pain relief in up to 80% of the cases [7, 13]. While this process does more than than only eliminate numbers of peripheral axons, it is consistent with the idea that hyperinnervation is a contributing factor in some chronically painful conditions. Additional studies are required to determine whether hyperinnervation persists following resolution of inflammation and how well this correlates with changes in sensitivity.

Acknowledgments

The authors wish to thank Michelle Winter for assistance with the rat behavioral assays. Funding for this piece of work was provided by NIH RO1HD049615 with core back up from NICHD P30HD002528.

References

ane. Aley KO, Messing RO, Mochly-Rosen D, Levine JD. Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci. 2000;20:4680–4685. [PMC free article] [PubMed] [Google Scholar]

2. Alfredson H, Ohberg 50, Forsgren S. Is vasculo-neural ingrowth the crusade of pain in chronic Achilles tendinosis? An investigation using ultrasonography and colour Doppler, immunohistochemistry, and diagnostic injections. Knee Surg Sports Traumatol Arthrosc. 2003;11:334–338. [PubMed] [Google Scholar]

three. Amann R, Schuligoi R. Inhibition of carrageenan-induced edema by indomethacin or sodium salicylate does not prevent the increment of nerve growth factorin the rat hind paw. Neurosci Lett. 2000;278:173–176. [PubMed] [Google Scholar]

four. Black JA, Liu S, Tanaka M, Cummins TR, Waxman SG. Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain. Pain. 2004;108:237–247. [PubMed] [Google Scholar]

5. Bohm-Starke N, Hilliges G, Falconer C, Rylander Due east. Increased intraepithelial innervation in women with vulvar vestibulitis syndrome. Gynecol Obstet Invest. 1998;46:256–260. [PubMed] [Google Scholar]

6. Bohm-Starke N, Hilliges M, Falconer C, Rylander Eastward. Neurochemical label of the vestibular nerves in women with vulvar vestibulitis syndrome. Gynecol Obstet Invest. 1999;48:270–275. [PubMed] [Google Scholar]

7. Bohm-Starke Northward, Rylander East. Surgery for localized, provoked vestibulodynia: a long-term follow-upward report. J ReprodMed. 2008;53:83–89. [PubMed] [Google Scholar]

8. Brown MF, Hukkanen MV, McCarthy ID, Redfern DR, Batten JJ, Crock HV, Hughes SP, Polak JM. Sensory and sympathetic innervation of the vertebral endplate in patients with degenerative disc disease. J Os Joint Surg Br. 1997;79:147–153. [PubMed] [Google Scholar]

9. Craft RM. Modulation of pain past estrogens. Hurting. 2007;132(Suppl 1):S3–12. [PubMed] [Google Scholar]

10. Di Sebastiano P, Fink T, Weihe E, Friess H, Beger HG, Buchler G. Changes of protein gene product ix.v (PGP ix.v) immunoreactive nerves in inflamed appendix. Dig Dis Sci. 1995;40:366–372. [PubMed] [Google Scholar]

eleven. Dib-Hajj SD, Fjell J, Cummins TR, Zheng Z, Fried Yard, LaMotte R, Blackness JA, Waxman SG. Plasticity of sodium channel expression in DRG neurons in the chronic constriction injury model of neuropathic hurting. Pain. 1999;83:591–600. [PubMed] [Google Scholar]

12. Garcia-Cosamalon J, Del Valle ME, Calavia MG, Garcia-Suarez O, Lopez-Muniz A, Otero J, Vega JA. Intervertebral disc, sensory nerves and neurotrophins: who is who in discogenic pain? J Anat. 2010 [PMC gratuitous article] [PubMed] [Google Scholar]

13. Gaunt Yard, Good A, Stanhope CR. Vestibulectomy for vulvar vestibulitis. J Reprod Med. 2003;48:591–595. [PubMed] [Google Scholar]

14. Gopinath P, Wan East, Holdcroft A, Facer P, Davis JB, Smith GD, Bountra C, Anand P. Increased capsaicin receptor TRPV1 in pare nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human being breast pain. BMC Womens Health. 2005;v:ii. [PMC gratuitous article] [PubMed] [Google Scholar]

xv. Grossmann L, Gorodetskaya N, Teliban A, Baron R, Janig W. Cutaneous afferent C-fibers regenerating along the distal nerve stump afterwards shell lesion show 2 types of cold sensitivity. Eur J Hurting. 2009;xiii:682–690. [PubMed] [Google Scholar]

sixteen. Guay J, Bateman Grand, Gordon R, Mancini J, Riendeau D. Carrageenan-induced hand edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1. J Biol Chem. 2004;279:24866–24872. [PubMed] [Google Scholar]

17. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Hurting. 1988;32:77–88. [PubMed] [Google Scholar]

eighteen. Janig West, Grossmann L, Gorodetskaya N. Mechano-and thermosensitivity of regenerating cutaneous afferent nerve fibers. Exp Brain Res. 2009;196:101–114. [PubMed] [Google Scholar]

19. Janse C, Peretz B, van der Roest M, Dubelaar EJ. Excitability and branching of neuroendocrine cells during reproductive senescence. Neurobiol Crumbling. 1999;20:675–683. [PubMed] [Google Scholar]

20. Kayser V, Guilbaud G. Local and remote modifications of nociceptive sensitivity during carrageenin-induced inflammation in the rat. Pain. 1987;28:99–107. [PubMed] [Google Scholar]

21. Li L, Hao JX, Fredholm BB, Schulte Thou, Wiesenfeld-Hallin Z, Xu XJ. Peripheral adenosine A(2A) receptors are involved in carrageenan-induced mechanical hyperalgesia in mice. Neuroscience. 2010 [PubMed] [Google Scholar]

22. Marchand F, Perretti M, McMahon SB. Role of the allowed system in chronic pain. Nat Rev Neurosci. 2005;six:521–532. [PubMed] [Google Scholar]

23. McMahon South, Koltzenburg Thousand. The changing role of primary afferent neurones in pain. Pain. 1990;43:269–272. [PubMed] [Google Scholar]

24. Miller RJ, Jung H, Bhangoo SK, White FA. Cytokine and chemokine regulation of sensory neuron function. Handb Exp Pharmacol. 2009:417–449. [PMC free article] [PubMed] [Google Scholar]

25. Morris CJ. Carrageenan-induced paw edema in the rat and mouse. Methods Mol Biol. 2003;225:115–121. [PubMed] [Google Scholar]

26. Rice FL, Albers KM, Davis BM, Silos-Santiago I, Wilkinson GA, LeMaster AM, Ernfors P, Smeyne RJ, Aldskogius H, Phillips HS, Barbacid K, DeChiara TM, Yancopoulos GD, Dunne CE, Fundin BT. Differential dependency of unmyelinated and A delta epidermal and upper dermal innervation on neurotrophins, trk receptors, and p75LNGFR. Dev Biol. 1998;198:57–81. [PubMed] [Google Scholar]

27. Riley JL, tertiary, Robinson ME, Wise EA, Price DD. A meta-analytic review of pain perception beyond the menstrual cycle. Pain. 1999;81:225–235. [PubMed] [Google Scholar]

28. Sanchis-Alfonso V, Rosello-Sastre Due east. Immunohistochemical analysis for neural markers of the lateral retinaculum in patients with isolated symptomatic patellofemoral malalignment. A neuroanatomic ground for anterior articulatio genus hurting in the active young patient. Am J Sports Med. 2000;28:725–731. [PubMed] [Google Scholar]

29. Sanchis-Alfonso V, Rosello-Sastre Due east, Monteagudo-Castro C, Esquerdo J. Quantitative assay of nerve changes in the lateral retinaculum in patients with isolated symptomatic patellofemoral malalignment. A preliminary study. Am J Sports Med. 1998;26:703–709. [PubMed] [Google Scholar]

thirty. Schubert TE, Weidler C, Lerch Chiliad, Hofstadter F, Straub RH. Achilles tendinosis is associated with sprouting of substance P positive nerve fibres. Ann Rheum Dis. 2005;64:1083–1086. [PMC costless commodity] [PubMed] [Google Scholar]

31. Tabo E, Eisele JH, Jr, Carstens Due east. Forcefulness of limb withdrawals elicited past graded noxious oestrus compared with other behavioral measures of carrageenan-induced hyperalgesia and allodynia. J Neurosci Methods. 1998;81:139–149. [PubMed] [Google Scholar]

32. Tall JM, Crisp T. Effects of gender and gonadal hormones on nociceptive responses to intraplantar carrageenan in the rat. Neurosci Lett. 2004;354:239–241. [PubMed] [Google Scholar]

33. Treede RD, Meyer RA, Raja SN, Campbell JN. Peripheral and primal mechanisms of cutaneous hyperalgesia. Prog Neurobiol. 1992;38:397–421. [PubMed] [Google Scholar]

34. Tympanidis P, Terenghi Chiliad, Dowd P. Increased innervation of the vulval vestibule in patients with vulvodynia. Br J Dermatol. 2003;148:1021–1027. [PubMed] [Google Scholar]

35. Wang G, Tokushige North, Russell P, Dubinovsky S, Markham R, Fraser IS. Hyperinnervation in abdominal deep infiltrating endometriosis. J Minim Invasive Gynecol. 2009;16:713–719. [PubMed] [Google Scholar]

36. Wilkinson KD, Lee KM, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J. The neuron-specific poly peptide PGP nine.five is a ubiquitin carboxyl-terminal hydrolase. Science. 1989;246:670–673. [PubMed] [Google Scholar]

37. Wintertime CA, Risley EA, Nuss GW. Carrageenin-induced edema in hind paw of the rat as an assay for antiiflammatory drugs. Proc Soc Exp Biol Med. 1962;111:544–547. [PubMed] [Google Scholar]

38. Woolf CJ. Generation of acute pain: key mechanisms. Br Med Bull. 1991;47:523–533. [PubMed] [Google Scholar]

39. Woolf CJ, Ma Q. Nociceptors--noxious stimulus detectors. Neuron. 2007;55:353–364. [PubMed] [Google Scholar]

xl. Woolf CJ, Thompson SW. The induction and maintenance of key sensitization is dependent on N-methyl-D-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states. Pain. 1991;44:293–299. [PubMed] [Google Scholar]

41. Zimmermann K, Herdegen T. Plasticity of the nervous system at the systematic, cellular and molecular levels: a mechanism of chronic pain and hyperalgesia. Prog Encephalon Res. 1996;110:233–259. [PubMed] [Google Scholar]