Hypersensitivity and hyperinnervation of the rat hind paw following carrageenan-induced inflammation

Studies of human tissue show that many chronic pain 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 present in these conditions, or other factors such as infection or extensive tissue damage. In the present study, we used a well established model of inflammation to examine cutaneous innervation density in relation to mechanical and thermal hypersensitivity. Adult female rats were ovariectomized to eliminate fluctuations in female reproductive hormones and one week later, a hind paw 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 by increased mechanical sensitivity of the contralateral paw at 72 h. Analysis of innervation density using PGP9.5 as a pan-neuronal marker at 72 h showed that inflammation resulted in a 2-fold increase in cutaneous innervation density. We conclude that inflammation alone is sufficient to induce sprouting of sensory cutaneous axon endings leading local tissue hyperinnervation, which may contribute to hypersensitivity that occurs in painful inflammatory conditions.     

Hypersensitivity and hyperinnervation of the rat hind paw following carrageenan-induced inflammation

Transcranial direct current stimulation (tDCS) has been found to produce significant changes in behavior, including a large increase of learning and performance for a difficult visual perceptual task (Clark et al., NeuroImage 2010). The mechanisms by which tDCS produces these behavioral effects are currently uncertain. One hypothesis is that anodal tDCS leads to increased metabolic activity in the brain, which enhances cognitive and memory processes. Here we examined the neuronal mechanisms by which tDCS influences learning by measuring changes in brain metabolite concentrations using proton magnetic resonance spectroscopy (1H MRS). As perception and learning can also influence neurochemistry, here we applied tDCS during rest. MRS data was obtained before and after 2.0 mA of anodal tDCS was applied for 30 min over electrode site P4, with the cathode placed on the contralateral arm. MRS data were acquired from the right parietal lobe beneath the anodal tDCS electrode, and from the homologous regions of the left hemisphere once before and once after tDCS. Significantly higher combined glutamate and glutamine levels were found in right parietal cortex, beneath the stimulating electrode, with non-significant increases in homologous regions of the opposite hemisphere. In addition, a significant interaction between hemispheres was found for tDCS effects on tNAA. These results suggest that changes in glutamatergic activity and tNAA may be related to the mechanisms by which tDCS influences learning and behavior.     

Characteristic features of actinomycin D-induced paw inflammation of the rat.

Features of paw edema induced by subplantar injection of actinomycin D (act D) were investigated in rats. The paw edema was produced as early as the 1st day and reached a maximal level on the 3rd or the 4th day. Thereafter, it began to subside progressively and was considerably reduced by the 16th day following act D (20 μgm) injection. A direct dose response relationship between the amount of act D injected and the intensity of the paw edema was obtained. No difference in β-glucuronidase and acid phosphatase activity was found between saline and act D-injected paws on the 2nd day. This was followed by an increase in the activity of both enzymes on the 4th, 8th, and 16th days after injection. The histamine content of the saline and act D-injected paws remained unchanged during the early phase of inflammation. A marked increase in the histamine content was noted during the late phase in the drug-injected paw. The effects of act D treatment on capillary permeability to Evans blue dye (EBD) and the edema formation of the paw revealed that a maximal increase in vascular permeability to EBD occurred on the 1st day and was maintained until the 8th day. In contrast to permeability, the paw edema on the 1st day was minimal and increased progressively until the 3rd or 4th day. Thereafter, both the permeability and the paw edema began to diminish and were considerably reduced on the 16th day. Aspirin and hydrocortisone treatment were ineffective in suppressing the act D-induced paw inflammation. Indomethacin produced a somewhat dose-related anti-inflammatory effect against the inflammation caused by this drug.     

Effect of chemical sympathectomy on cardiac hypertrophy and hemodynamics following myocardial infarction in the rat

The effects of sympathectomy on cardiac structure and function were studied in an animal model of myocardial infarction. Ninety-six rats were double randomized to control or infarction disease state and to placebo or chemical sympathectomy (guanethidine, 30 mg/kg daily, intraperitoneal). Five weeks after anterior infarction, there was hypertrophy in placebo-treated animals in myocardial fibers remote from the infarct (9.8 +/- 1.8 microns in infarction vs 8.1 +/- 1.0 microns in control, p less than 0.05). However, myocardial hypertrophy was not present in guanethidine-treated animals (8.6 + 1.6 microns in infarction vs 8.0 + 0.6 microns in control, p = N.S.). Guanethidine treatment caused significant reductions in systolic arterial blood pressure and indices of left ventricular contractility and relaxation (p less than 0.05), but these effects were not different between infarct and control treatments (p = N.S.). Although both effects of guanethidine treatment (sympathectomy and hemodynamic) were correlated with myocardial fiber diameter by univariate analysis, only sympathectomy was significant by stepwise regression analysis (p less than 0.05). Therefore, the cardiac sympathetic nerves have important effects on the development of hypertrophy after myocardial infarction, and sympathectomy alters this process in the rat model.     

Neurogenic inflammation in airways.

Neurogenic inflammation, due to release of neuropeptides from sensory nerves, has been demonstrated in airways of several species, particularly rodents, and may contribute to the inflammatory response in asthmatic airways. Tachykinins (substance P and neurokinin A) and calcitonin-gene-related peptide released from airway sensory nerves may cause bronchoconstriction, vasodilatation, plasma exudation and mucus secretion. Sensory nerves may become sensitised by inflammatory products and triggered by mediators such as bradykinin, resulting in exaggerated inflammation. The effects of tachykinins may be further amplified by loss of the major degrading enzyme, neutral endopeptidase, from epithelial cells. Several strategies for reducing neurogenic inflammation are possible.     

Relationship between oedema and plasma exudation in rat paw carrageenin inflammation

High (500 g) and low 100g) doses of carrageenin produced different temporal extravasation of plasma protein when injected into the rat paw. High doses caused a continuous extravasation parallel to the oedema increase. With low doses, main exudation occurred during the first 2 h and was minimal when the oedema was maximal (4th hour). Pre-treatment with indomethacin (2 mg/kg/i.p.) was effective in reducing oedema and protein extravasation for both doses of carrageenin. Indomethacin given 2 h after carrageenin had no effect upon the oedema caused by the low doses, but was effective with the high doses of carrageenin. The antioedematogenic effect of a non-steroid anti-inflammatory agent, such as indomethacin, was related to its effect upon plasma exudation, i.e. the agent was effective when there was a correlation between increment of oedema and plasma extravasation. The continuously increased plasma extravasation indicated the continuous presence of an active damaging stimulus. Our results explain several apparently contradictory observations reported in the literature.     

Neurogenic inflammation in the rat trachea. I. Changes in venules, leucocytes and epithelial cells

Summary This study was done to characterize the morphological changes in the respiratory mucosa that occur in neurogenic inflammation, which is a type of inflammation mediated by substances released from sensory nerves. Neurogenic inflammation was produced in the trachea and bronchi of atropine-treated Long-Evans rats by electrically stimulating the left or right superior laryngeal and vagus nerves. This procedure is known to increase vascular permeability in the airways, presumably as a consequence of antidromic activation of sensory vagal axons (Lundberg & Saria, 1982). By using a particulate tracer (Monastral blue, 30 mg kg^–1 i.v.) that does not cross the walls of normal tracheal blood vessels but does cross the endothelium of abnormally permeable vessels, it was possible to identify which blood vessels were affected in neurogenic inflammation. Light and electron microscopic examination of tracheas prepared after 2 or 5 min of vagal stimulation revealed that postcapillary venules and collecting venules 7–80 (m in diameter were labelled by extravasated Monastral blue but capillaries, arterioles, and larger venules were not. Venules from which the extravasation occurred had gaps as wide as 1.5 m between endothelial cells. Most of the abnormally permeable venules were located just beneath the airway epithelium in regions between the cartilaginous rings. Extravasation also occurred from venules in the mucosa overlying the posterior membrane of the extrathoracic trachea, but little occurred in the posterior membrane of the intrathoracic trachea. After unilateral vagal stimulation, vascular permeability was increased on both sides of the trachea; it was also increased in first through fourth order bronchi but only on the side of stimulation. Leucocytes (principally monocytes and neutrophils) were adherent to the endothelium of some of the abnormally permeable venules. Erythrocytes and platelets also were adherent to the walls of some venules. These changes in venules were accompanied by a degranulation of epithelial secretory cells, widening of the spaces between tracheal epithelial cells, and probably an increase in epithelial permeability. Neurogenic inflammation in the trachea and bronchi of rats is thus characterized by increased permeability of postcapillary venules and collecting venules in specific regions of the respiratory mucosa as well as adherence of leucocytes, erythrocytes and platelets to the endothelium of these venules and prominent changes in the respiratory epithelium.     

Degeneration secretion of saliva in the rat following sympathectomy

1. After previous extirpation of the superior cervical ganglion saliva was found to flow from the cannulated submaxillary duct of rats under chloralose anaesthesia. The secretion started 13-14.5 hr after sympathectomy, increased gradually in rate and then slowed down and ceased, lasting for about 12 hr.2. Injection of alpha- or beta-adrenoreceptor blocking drugs reduced the flow and a combination of both types of drugs abolished it. It is concluded that the secretion was caused by noradrenaline released from the degenerating nerves, and that the phenomenon is an example of the degeneration activity described in other preparations as an effect of section of post-ganglionic autonomic nerves.3. A similar, but less pronounced, secretion was found in the parotid gland of the rat.     

Interaction between nitric oxide and substance P on heat-induced inflammation in rat paw

To elucidate the interaction between nitric oxide (NO) and substance P (SP) in neurogenic inflammatory responses, we measured the change in the degree of Evans blue leakage and NO levels in perfusate from the subcutaneous space in the rat instep following noxious heat stimulation (47 degrees C for 30 min). Furthermore, the effects of drugs affecting nitric oxide synthase were examined. Noxious heat stimulation caused on an increase in NOx, or NO2- and NO3- into the perfusate in parallel with plasma extravasation. Nw-nitro-L-arginine methylester (L-NAME: 100 mg/kg once daily.) intraperitoneally (i.p.) given five times (chronic treatment) significantly suppressed the increase in Evans blue extravasation induced by heat stimulation, whereas acute treatments with L- and D-NAME (100 mg/kg once, i.p.) did not show any significant effect. NO release induced by heating also was significantly suppressed by chronic pretreatment with L-NAME, but not by acute treatment. SP (10(-5) M) applied into the perfusate caused a remarkable increase in the NOx release into the perfusate. Intra-arterial injection of RP67580 (1 mg/kg) on the perfused side, but not SR48968 (1 mg/kg), significantly attenuated the increases in Evans blue leakage and NOx release during heat stimulation. These results suggest that heat-induced SP release from the peripheral endings of small-diameter afferent fibers causes NO generation through NK-1R, and that this gas act to elicit or enhance inflammatory responses.     

Neurogenic inflammation in the rat trachea. II. Identity and distribution of nerves mediating the increase in vascular permeability

Summary This study addresses the question of whether increased vascular permeability, which is a prominent feature of neurogenic inflammation in the respiratory tract, is mediated by sensory axons that end near venules in the airway mucosa. In these experiments, neurogenic inflammation was produced in the tracheal and bronchial mucosa of atropine-treated Long-Evans rats by electrical stimulation of the left or right superior laryngeal nerve and/or cervical vagus nerve. The particulate tracer Monastral blue was injected intravenously to localize the sites of increased vascular permeability, and microspectrophotometry was used to measure the amount of extravasated Monastral blue in the trachea and thereby quantify the increase in vascular permeability. In some rats, selective denervations were made to locate the cell bodies of neurons that mediate the increase in vascular permeability; in others, fluorescence immunohistochemistry and quantitative electron microscopic methods were used to determine which structures in the tracheal mucosa are innervated by these neurons. The study revealed that the vagally mediated increase in vascular permeability was sudden, transient (half-life=2.4 min) and restricted to venules. Stimulation of the left or right superior laryngeal nerve increased the permeability of venules in the extrathoracic trachea, whereas stimulation of either vagus nerve increased vascular permeability in the intrathoracic trachea and bronchi. All nerves had bilateral effects in the trachea, but the vagus nerves had largely unilateral effects in the bronchi. Neurons that mediated the increase in venular permeability had their cell bodies in the jugular (superior sensory) ganglion of the vagus nerve or rostral portion of the nodose (inferior sensory) ganglion. Preganglionic autonomic vagal neurons in the brain stem were not essential for this increase in venular permeability. Few nerves identifiable by substance P-immunohistochemistry or electron microscopy were located near the affected venules, and no nerves were within 1 m of the walls of venules. However, the epithelium and arterioles of the airway mucosa were densely innervated. All intraepithelial nerves were within 0.1 m of epithelial cells, and at least two-thirds of nerves near arterioles were within 1 m of the vessel walls. We conclude that the increase in venular permeability associated with neurogenic inflammation in the trachea and bronchi of rats is mediated by sensory axons that travel in the vagus nerves and superior laryngeal nerves. We question whether tachykinins from the sensory nerves mediate the increase in vascular permeability through a direct action on venules, and raise the possibility that these nerves evoke the release from epithelial cells of mediators that contribute to the increase in vascular permeability.     

Neurogenic inflammation in skin blisters

Summary The histology of blisters induced in rats using dry-ice or vacuum applied to the hind limb footpad has been examined by light microscopy and indirect fluorescence immunohistochemistry for substance P like immunoreactivity. The effects of sciatic nerve stimulation on plasma extravasation as determined by estimation of Evans Blue content of blister fluid has been examined. Plasma extravasation occurs in both blister types following nerve stimulation. Plasma extravasation may be induced by substance P released from nerve terminals in the blister wall. Vacuum induced blisters have less tissue and nerve damage and therefore less of an inflammatory response, but plasma extravasation is greater in thermal blisters. Blisters serve as a useful model for the study of neurogenic inflammation.     

Secretory effects of 5-hydroxytryptamine following neonatal sympathectomy in rat parotid gland.

Unilateral sympathetic denervation of rat parotid glands was performed within 4 h after birth. Nine weeks later the glands were used for in-vitro studies of amylase secretion, and 86Rb+ was used as a marker for potassium efflux. The non-denervated contralateral glands served as controls. The tissue concentrations of 5-hydroxytryptamine and its metabolite 5-hydroxyindole acetic acid were also measured. 5-Hydroxytryptamine caused a significant dose-dependent increase in amylase secretion, which was inhibited by methysergide. There was no difference between controls and denervated glands. 5-Hydroxytryptamine was without effect on potassium efflux from either denervated or control glands. The sympathectomy caused increased levels of 5-hydroxytryptamine and 5-hydroxyindole acetic acid as compared with contralateral controls. The results suggest that 5-hydroxytryptamine influences the two main secretory processes in rat parotid gland differently. A significant amylase discharge was seen following 5-hydroxytryptamine stimulation, whereas no effect was seen on 86Rb+ efflux. Although it is also proposed that there are no 5-hydroxytryptamine-associated nerves in the superior cervical ganglion innervating parotid tissue, it seems that there is a complex connection between the sympathetic pathway and the serotoninergic system.     

Effects of acute inflammation induced in the rat paw on the deep digital flexor tendon

The tendon is commonly affected by inflammation, and in such situations, the tissue undergoes a process of reorganization of the extracellular matrix to improve and regenerate the affected region. Little is known about the mechanisms that trigger inflammation in the tissues surrounding the affected area. The objective of this study was to biochemically and morphologically analyze the deep digital flexor tendon at the peak of acute inflammation in the rat paw. Wistar rats were divided into the following three groups: those that received injection of 1% carrageenan, those that received 0.9% NaCl, and those that received nothing. The deep digital flexor tendon was divided into the distal, proximal, and intermediate regions. For biochemical analysis, the tendons were treated with guanidine hydrochloride and analyzed by sodium dodecyl sulfate-polyacrilamide gel electrophoresis. Proteins, glycosaminoglycans (GAGs), and hydroxyproline were quantified, and metalloproteinases were analyzed. The GAGs were analyzed by agarose gel electrophoresis. Tissue sections were stained with hematoxylin-eosin, toluidine blue, and Ponceau SS. The content of proteins and GAGs was smaller in the group receiving the application of carrageenan. The concentration of hydroxyproline in the two tendon regions that respond to tension forces was higher in the inflammation group. The metalloproteinase-9 was detected in the distal region, and a thicker epitenon with cellular infiltrate was observed in the groups with inflamed paws. Meanwhile, a better organization of collagen bundles was observed in the two tension regions of that same group. Our results show that although the tendon was not directly inflamed, changes in the surrounding structural and biochemical parameters were observed.     

Neurogenic inflammation in the skin of the rabbit

A flare-like spread of vasodilatation can be demonstrated in the limb skin of the anaesthetised rabbit using laser doppler flowmetry. Spread of dilatation from a small mechanical injury (nicking the skin) extends 5–10 mm. The size and time course of the dilatation are as expected from the low frequency C-nociceptor firing that occurs in response to similar nearby injury. Since C-nociceptors cause significant vasodilator responses at frequencies less than 1 Hz, it is proposed that they may often have a proinflammatory action without producing any reflex or sensory effects.     

Neurogenic inflammation caused by wool fabric in the rat; possible mediation by substance P

We have measured plasma extravasation in response to stimulation of the rat hind footpad with wool fabric. The stimulus was delivered using a constantly weighted disc of wool rotating at 4 revolutions per minute for 30 min on the base of blisters induced by vacuum. The protein content of prestimulated blister base perfusate was 38 +/- 2 micrograms/ml in a 30-min collection. With stimulation this increased to 80 +/- 10 micrograms/ml (P less than 0.001; Student's paired t-test). No increase in plasma extravasation was observed in adult animals pretreated as neonates with capsaicin indicating that the plasma extravasation observed was neurogenically mediated. Plasma extravasation was also observed with substance P added to the perfusate. The threshold for substance P-induced plasma extravasation was 2 X 10(-11) mol/30 min. These results suggest that neurogenic inflammation induced by the mechanical stimulus of wool fabric may be mediated by substance P. This reaction may be involved in wool-induced prickle and itch noted in some individuals.     

Neurogenic inflammation,cell adhesion molecules and future trends in inflammation therapy

Inflammation Research -