Different Desensitization Patterns for Sensory and Vascular TRPV1 Populations in the Rat: Expression, Localization and Functional Consequences

Background and purpose TRPV1 is expressed in sensory neurons and vascular smooth muscle cells, contributing to both pain perception and tissue blood distribution. Local desensitization of TRPV1 in sensory neurons by prolonged, high dose stimulation is re-engaged in clinical practice to achieve analgesia, but the effects of such treatments on the vascular TRPV1 are not known. Experimental approach Newborn rats were injected with capsaicin for five days. Sensory activation was measured by eye wiping tests and plasma extravasation. Isolated, pressurized skeletal muscle arterioles were used to characterize TRPV1 mediated vascular responses, while expression of TRPV1 was detected by immunohistochemistry. Key results Capsaicin evoked sensory responses, such as eye wiping (3.6±2.5 versus 15.5±1.4 wipes, p<0.01) or plasma extravasation (evans blue accumulation 10±3 versus 33±7 µg/g, p<0.05) were reduced in desensitized rats. In accordance, the number of TRPV1 positive sensory neurons in the dorsal root ganglia was also decreased. However, TRPV1 expression in smooth muscle cells was not affected by the treatment. There were no differences in the diameter (192±27 versus 194±8 µm), endothelium mediated dilations (evoked by acetylcholine), norepinephrine mediated constrictions, myogenic response and in the capsaicin evoked constrictions of arterioles isolated from skeletal muscle. Conclusion and implications Systemic capsaicin treatment of juvenile rats evokes anatomical and functional disappearance of the TRPV1-expressing neuronal cells but does not affect the TRPV1-expressing cells of the arterioles, implicating different effects of TRPV1 stimulation on the viability of these cell types.


Introduction
The therapeutic potential of TRPV1 modulation in various painful states has been recognized in a series of publications some 40 years ago [1][2][3][4]. In these early works capsaicin specific responses were related to a specific receptor, which could be desensitized by the application of high dose of capsaicin at an early phase of the life of the rat. These robust changes in sensory function led to the recognition of pharmacological approach to manipulate nociceptors [5]. Capsaicin desensitization therefore become a powerful approach to characterize peripheral nociceptors and to achieve topical analgesia. Although molecular identification of the receptor responsible for capsaicin mediated responses [6] resulted in a rise in therapeutic interest in systemic TRPV1 modulation to inhibit the pain pathway, no successful antagonists were introduced to the market [7], probably due to the unfavorable side effects of the systemic treatment [8]. On the other hand, local capsaicin desensitization recently re-emerged as a therapeutic method to selectively defunctionalize capsaicin expressing sensory neurons and to achieve local analgesia [9,10].
In contrast with the acute desensitization, neonatal capsaicin treatment results in the deletion of TRPV1 expressing peripheral sensory neurons [19,20]. Capsaicin evoked sensory neuronal cell death may be mediated by Ca 2+ overload of the TRPV1 expressing cells [21].
Activation of TRPV1 leads to central (pain) and to local ''sensory-efferent'' effects [22]. These include the release of vasoactive agents (such as calcitonin gene-related peptide) from sensory neurons and subsequent vasorelaxation [23]. In recent years, TRPV1 expression has been identified in many cells types in addition to sensory neurons as referenced by Keeble et al in recent reviews [24,25]. In particular, TRPV1 expression was detected in various cell types in the brain [26], and in arteriolar smooth muscle [27]. In accordance with this latter non-neuronal localization, activation of TRPV1 resulted in a substantial vasoconstriction in vivo and in vitro [27,28] through direct activation of endogenous TRPV1 in vascular smooth muscle cells [29].
The effects of capsaicin desensitization were investigated here. Capsaicin desensitization is the only clinically available, approved and effective treatment option to regulate TRPV1 mediated sensory functions. Since functional TRPV1 expression has been identified in non-neuronal cells, these receptors may have physiological functions which may also be modulated by therapeutic capsaicin applications. Morphological and functional studies were performed to reveal differences between desensitization of sensory neurons and vascular smooth muscle cells. Our data suggest that capsaicin desensitization has only a transient effect on the vascular TRPV1. Therefore, vascular TRPV1 function is maintained even if the same treatment depletes sensory neurons.

Capsaicin pretreatment of rats
Newborn rats (at day 14 of life) were pretreated with Diaphyllin (Richter, Hungary), Bricanyl (Astra Zeneca, Hungary), and atropine (Egis, Hungary, 100 g/0,1 ml i.p.). Ten minutes later animals were injected with capsaicin (subcutaneously). The procedure was repeated for a total of five consecutive days. The total dose of capsaicin was 300 mg/kg, administered on a dose schedule of 10 mg/kg, 20 mg/kg; 50 mg/kg; 100 mg/kg; and 120 mg/kg on days 1 through 5, respectively. Rats were then kept in the animal facility for 10 weeks when experiments were performed. The weight of the rats was in the range of 360-492 g at this time.

Measurement of capsaicin evoked sensory irritation
One drop (10 ml) of capsaicin solution (50 mg/ml in physiological saline) was put into the right or left conjunctiva of the rat, in a random order. The number of eye wipes was counted during 60 s. Rats were scarified after capsaicin treatments.

Measurement of capsaicin evoked involuntary sensory neuronal functions
Plasma extravasation was measured according to Pinter et al. [30]. Rats were anesthetised and a tail vein was cannulated for the injection of Evans blue dye (30 mg/kg). One min after Evans blue administration capsaicin was injected (1 mg/kg, i.v.). Rats was sacrificed by transcardiac perfusion with 50 ml of 0.9% w/v saline, at 37uC, through the left cardiac ventricle 10 min after injection of Evans blue. The urinary bladder was removed and weighed, and the Evans blue was extracted in 1 ml of formamide for 24 h. Evans blue content was determined by spectrophotometry (at 620 nm). Plasma extravasation was expressed as the content of Evans blue dye in micrograms per gram of wet tissue.

Preparation of cannulated skeletal muscle arterioles
Isolation of the skeletal (gracilis) muscle arterioles of the rat and diameter measurement of the arterioles were performed as described earlier [12]. The internal diameters of the cannulated skeletal muscle (m. gracilis) arterioles were determined at the midpoint of the arteriolar segment by videomicroscopy. Cannulated arterioles were incubated in a physiological solution (PSS, composition in mM: 110 NaCl, 5.0 KCl, 2.5 CaCl 2 , 1.0 MgSO 4 , 1.0 KH 2 PO 4 , 5.0 glucose and 24.0 NaHCO 3 equilibrated with a gas mixture of 10% O 2 and 5% CO 2 , 85% N 2 , at pH 7.4.). Experiments were started after the development of a spontaneous tone in response to intraluminal pressure of 80 mmHg. First, acetylcholine (1 nM-10 mM) was used to determine dilatative capacity (acetylcholine causes endothelium dependent vasodilatation), and then norepinephrine (1 nM-10 mM) was applied to measure maximal constrictive response (norepinephrine causes smooth muscle dependent constriction). Vascular autoregulation (myogenic response) were also determined. Intraluminal pressure was increased from 20 to 120 mmHg in 20 mmHg increments and arteriolar diameter was measured after 4 min incubations at each intraluminal pressures. These measurements were performed in PSS to assess the active myogenic response. Passive diameter was determined in Ca 2+ free PSS at the end of the experiments. Changes in diameter in response to a TRPV1 agonist was tested with cumulative dose (capsaicin, 1 nM-30 mM).

Materials and solutions
Chemicals were from Sigma-Aldrich (St. Louis, MO, USA). Capsaicin (8-Methyl-N-vanillyl-trans-6-nonenamide) was dissolved in ethanol. Norepinephrine and acetylcholine were dissolved in distilled water. Evans Blue concentration was measured by using a NOVOstar plate reader.

Data analysis and statistical procedures
Arteriolar diameter was determined by measuring the distances between the intraluminal sides of the arteriolar wall (inner diameter). Data are shown as average diameter 6 S.E.M. Statistical analysis was made by Microsoft Excel using Student's t-tests. P-values,0.05 were considered to be significant.

Effects of desensitization on capsaicin-evoked eyewiping movements
Sensory irritation was assessed by eye wiping assays. Capsaicin evoked a high number of wiping movements in control rats (15.561.4 wiping movements, n = 10), while desensitized rats (neonatal capsaicin application) were almost insensitive to the same treatment (3.662.5 wipes, n = 10; p,0,001, Fig. 1A).

Effects of desensitization on capsaicin -induced plasma extravasation
Systemic activation of TRPV1 expressing sensory neurons by capsaicin resulted in an increase in Evans blue accumulation in the urinary bladder of control rats (3367 mg/g wet tissue; n = 9, Fig. 1B). Neonatal capsaicin desensitization significantly attenuated this response (Evans blue accumulation decreased to 1063 mg/ g wet tissue; n = 5, p = 0,03 versus control, Fig. 1B).

Immunochemistry of TRPV1
Dorsal root ganglia (DRG) were isolated from control and desensitized rats and TRPV1 expression was evaluated by immunohistochemistry. TRPV1 expression was found in a subset of neurons (neurons were visualized by neurofilament specific staining) in control rats but was missing in desensitized rats (Fig. 2). However, TRPV1 like immunostaining was also found in blood vessels associated with the DRGs, irrespective to the desensitization procedure. The localization of that TRPV1 expression to vascular smooth muscle cells was tested by the co-application of antisera against TRPV1 and vascular smooth muscle actin (Fig. 3). Indeed, some of the TRPV1 immunoreactivity overlapped with vascular smooth muscle specific staining. We thus find that, that while neonatal capsaicin treatment was found to be effective to eliminate TRPV1 immunoreactivity in sensory neurons, it was without effects on the vascular smooth muscle cell located TRPV1 immunoreactivity (Fig. 3). Similarly, TRPV1 expression was not affected by neonatal capsaicin treatment in the gracilis muscle of the rat (Fig. 4).

Physiological effects of neonatal capsaicin desensitization on isolated skeletal muscle arterioles
There were no significant differences between control and desensitized rats in acetylcholine mediated dilation (Fig. 5A), norepinephrine mediated constriction (Fig. 5B) and vascular autoregulation (spontaneous myogenic response, Fig. 5C) in isolated skeletal muscle resistance arteries. Moreover, capsaicin mediated arteriolar constrictions were also unaffected in both groups of rats (Fig. 5D), which was in contrast with the capsaicin mediated sensory functions (Fig. 1).
Direct application of capsaicin (1 mmol/L) resulted in a transient constriction (maximal decrease of arteriolar diameter was 7063% at 105 s) followed by a dilation in the continuous presence of capsaicin (constriction at the end of the 20 min long incubation was only 866%) (Fig. 6A). Besides to the acute desensitization (desensitization in the presence of the agonist) tachyphylaxis (desensitization to the repeated application of the agonist) was also determined. Although tachyphylaxis was apparent upon the second capsaicin challenge (first versus second treatment on Fig. 6A), capsaicin treatment did not eliminate capsaicin responsiveness completely (maximal constriction upon Figure 2. Effects of neonatal capsaicin desensitization on sensory TRPV1 expression. TRPV1 expression was examined in the dorsal root ganglia of control and capsaicin desensitized (neonatal capsaicin treatment) adult rats. Tissue sections were probed with antibodies specific to TRPV1 (red) and neurofilament (green). TRPV1 expression was present in a subset of sensory neurons in control rats, which was missing in desensitized rats within the dorsal root ganglia. However, TRPV1 immunoreactivity was also found in non-neuronal cell types, located at the outer regions of ganglia (regions occupied by sensory neurons are indicated by the freehand drawing). Images are representative of at least 10 sections. doi:10.1371/journal.pone.0078184.g002 second capsaicin treatment was 2669% at 270 s, while constriction was only 1064% at the end of the 20 min long treatment, Fig. 6A). To test if capsaicin mediated desensitization may affect the viability of the different vascular cell types, smooth muscle (norepinephrine, Fig. 6B) and endothelium (acetylcholine, Fig. 6C) mediated responses were also tested. Norepinephrine mediated responses were almost identical (Fig. 6B), while the limited shift in acetylcholine responses (Fig. 6C) did not reach the level of significance (p.0.05), suggesting that acute in vitro TRPV1 desensitization did not have a significant effect of TRPV1 independent functions of skeletal muscle arterioles.

Discussion
Capsaicin containing herbs were used from the prehistoric era in the medicinal efforts to relieve pain and inflammation [31]. Capsaicin treatment was introduced to eliminate sensory responses about fifty years ago [3] and these experiments lead to the identification of primary nociceptors [5]. Cloning of the receptor (named as TRPV1) and the development of knock out models confirmed its role in inflammatory hyperalgesia [32]. It was even proposed that there are endogenous ligands (endovanilloids) binding to the capsaicin binding site of TRPV1 [23], although the physiological relevance of these proposed endovanilloids to modulate TRPV1 are still under debate, especially in skeletal muscle arterioles [33].
TRPV1 appeared to be a sensory neuronal specific receptor, which has a role in nociception and can be selectively targeted by small molecules. Not surprisingly, TRPV1 become a pharmaceutical target to develop a new generation of painkillers as well as to treat other therapeutic conditions [7]. Although almost all of the major pharmaceutical companies initiated a TRPV1 research program, these efforts have not yet produced approved drugs [34].
The apparent failure of introduction of TRPV1 antagonists into the clinical practice is probably not the result of insufficient potency of the developed antagonists [34]. Nonetheless, clinical application of the TRPV1 agonists capsaicin and resiniferatoxin offer an alternative to TRPV1 antagonists for treating challenging painful conditions such as neuropathic pain [10,35]. It is important to note that therapy to achieve desensitization is different from that based on the application of antagonists in terms of the potential side effects. Antagonists may cause side effects if their receptors play a significant physiological role, while desensitizing agonists may activate receptors which are expressed, but without much physiological role. Interestingly, while recent data suggested that TRPV1 may be expressed in other cell types than sensory neurons (see Keeble et al [24,25] for references and [36]), the potential side effects of capsaicin desensitization had not been tested on these receptors.
Selective elimination of TRPV1 positive sensory neurons and attenuation of capsaicin evoked sensory neuron mediated effects were found following systemic neonatal capsaicin treatment confirming earlier data [37,38]. Effects of this treatment on the vascular smooth muscle located TRPV1 was addressed here for the first time. It was found that neonatal capsaicin treatment does not affect arteriolar TRPV1 function and expression. It is in accordance with the limited if any permanent vascular effects of neonatal capsaicin desensitization.
It has been suggested that capsaicin has biphasic effects on the vasculature: at lower concentrations, capsaicin (up to 10 nM) evokes vasodilation in skin due to sensory nerve activation, while higher concentrations (0.1-1 mM) lead to substantial constrictions in skeletal muscle arterioles due to non-neuronal TRPV1 stimulation [27]. This apparent difference in sensitivity may be due to (i) receptor sensitivity or (ii) a difference in TRPV1 receptor density in skin and skeletal muscle arterioles as been concluded by Fernandes et al [25]. As the matter of the first hypothesis a recent study designed to detect pharmacological differences between TRPV1 populations in the sensory nerves and in the arteriolar smooth muscle proved that these TRPV1 populations are pharmacologically different: some TRPV1 agonists were irritative (a measure of sensory neuronal TRPV1 activation), but without vasoconstrictive effects [29]. Capsaicin evoked vasoconstriction was mediated by direct activation of arteriolar smooth muscle located TRPV1 and was missing in TRPV1 knock out mice, suggesting that both the irritation (sensory neuronal response) and vasoconstriction (smooth muscle response) are mediated by TRPV1 [29]. As the matter of the second hypothesis (different expression of TRPV1 in perivascular nerves) a clear tissue specific difference was described. Arteries in the skin were densely innervated by TRPV1 positive fibers, in contrast with arteries in the skeletal muscle [27]. This fact suggests that sensory neuron mediated dilation plays a dominant role in the skin upon TRPV1 stimulation, but may have a limited effect in the skeletal muscle.
TRPV1 was found to be important in inflammation affecting arteriolar diameter. The most prominent contribution of TRPV1 stimulation to the neurogenic inflammation is the local release of neuropeptides (such as CGRP and substance P) from the sensory neurons [23], which is missing in capsaicin desensitized rats [5]. However, it was found recently, that TRPV1 stimulation also increases the plasma concentration of PACAP-38, an antiinflammatory peptide [39], and somatostatin [40] suggesting a complex regulation of neurogenic inflammatory response, as summarized by Alawi and Keeble in an exemplary review [24]. These observations suggest that TRPV1 has a role in vascular inflammation. Chronic physiological activation of TRPV1 expressing sensory neurons may contribute to the inflammatory response and to painful effects such as hyperalgesia and allodynia [24]. In particular, diabetic rats showed increased TRPV1 function (while TRPV1 expression decreased) in the dorsal root ganglia [41], a feature which is similarly present in human diabetic patients [42]. This represents a local feedback regulating TRPV1 activity: TRPV1 activation causes a desensitization of the TRPV1 itself (limiting hyperalgesia and allodynia), which is mediated by decreased expression of TRPV1 in the tissues. This suggests that TRPV1 desensitization has an effect on tissue perfusion. In particular, diabetic or other kind of neuropathies result in a decrease in TRPV1 expressing sensory neurons, mediating vasodilation. As a result, patients with diabetes may have an impaired regulation of blood flow leading to the dominance of smooth muscle TRPV1 mediated vasoconstrictions over the physiological sensory neuronal TRPV1 mediated vasodilations. This altered vascular response may contribute to the progression of the disease in the skin. was tested by the contractile response developing in response to increasing intraluminal pressure (from 20 mmHg to 120 mmHg in 20 mmHg increments). Finally, capsaicin mediated responses were also tested by the cumulative application of the drug. Capsaicin evoked a dose-dependent constriction, supporting a physiological role for this receptor in vascular smooth muscle cells, which was not affected by the desensitization protocol (Panel D, symbols represent the mean 6 SEM of n = 4 determinations for both groups). doi:10.1371/journal.pone.0078184.g005 The apparent difference between sensory neurons and vascular smooth muscle cells in response to high capsaicin stimulation (desensitization) described in this work may be explained by at least two mechanisms. First, vascular smooth muscle cells are able to proliferate, therefore the seriously damaged TRPV1 expressing cells may be replaced. Alternatively, capsaicin damage is less severe in vascular smooth muscle cells compared to sensory neurons. To test this latter possibility, isolated arteries were treated with capsaicin in vitro. Arteriolar TRPV1 was rapidly (within minutes) desensitized to capsaicin and capsaicin treatment did not affect TRPV1 independent functions, suggesting that capsaicin mediated Ca 2+ toxicity did not occur in these cells.
We propose that vascular smooth muscle cells can be activated by the application of high doses of capsaicin, but the Ca 2+ toxicity is limited by fast desensitization, in accordance with previous findings [12]. Vascular smooth muscle cells are therefore protected from prolonged capsaicin treatments and maintain their viability.
These cells then are able to regenerate and regain capsaicin responsiveness (and probably their physiological role) within a short period of time. In contrast, sensory neurons seem to be eliminated and not replaced upon neonatal capsaicin treatment. Taken together, our data suggest that capsaicin desensitization is an effective method to specifically regulate sensory functions, in contrast with TRPV1 antagonists, which may affect vascular TRPV1 mediated processes in addition to sensory functions. Figure 6. Functional effects of acute capsaicin desensitization on vascular TRPV1. The short-term effects of capsaicin were tested here. Arterioles were isolated from control rats and were treated with capsaicin (1 mM, First treatment). Capsaicin was washed away after the 20 min long treatment and the arterioles were incubated in the physiological buffer alone for 40 min (regeneration). Then capsaicin effects we re-tested (Second treatment) to estimate the level of tachyphylaxis (n = 6 arteries were tested, symbols represent the mean 6 SEM of time-matched arteriolar diameter). Acute desensitization to capsaicin was apparent upon the first treatment (decrease and then increase in arteriolar diameter in the continuous presence of capsaicin, maximal decrease was 7063% at 95 s after capsaicin application, Panel A). The magnitude of the response was decreased upon the second capsaicin treatment on the same arterioles (tachyphylaxis, decreased response to repeated capsaicin stimuli, maximal decrease was 2769% at 125 s after capsaicin application, Panel A). This acute capsaicin desensitization (20 min treatment) did not affect norepinephrine mediated constrictions (symbols represent the mean 6 SEM of n = 6 determinations, Panel B) or acetylcholine mediated dilations (symbols represent the mean 6 SEM, n = 6, Panel C). doi:10.1371/journal.pone.0078184.g006 Desensitization of Vascular TRPV1 PLOS ONE | www.plosone.org