Cellular localization of kinin B1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nα-Bodipy]-des-Arg9-bradykinin

Background The kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B1R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nα-Bodipy]-des-Arg9-BK (BdABK) and selective antibodies. Methods Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.). Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography. The B1R selectivity of BdABK was determined by assessing its ability to displace B1R [125I]-HPP-desArg10-Hoe140 and B2R [125I]-HPP-Hoe 140 radioligands. The in vivo activity of BdABK was also evaluated on thermal hyperalgesia. Results B1R was increased by 18-fold (mRNA) and 2.7-fold (binding sites) in the thoracic spinal cord of STZ-treated rats when compared to control. BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM). In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively. Intraperitoneal BdABK and des-Arg9-BK elicited dose-dependent thermal hyperalgesia in STZ-treated rats but not in control rats. The B1R fluorescent agonist was co-localized with immunomarkers of microglia, astrocytes and sensory C fibers in the spinal cord of STZ-treated rats. Conclusion The induction and up-regulation of B1R in glial and sensory cells of the spinal cord in STZ-diabetic rats reinforce the idea that kinin B1R is an important target for drug development in pain processes.


Background
Kinins are vasoactive peptides and central mediators acting through the activation of two G-protein-coupled receptors (R) denoted as B 1 and B 2 [1,2]. The B 2 R is widely and constitutively expressed in central and peripheral tissues and is activated by its preferential agonists bradykinin (BK) and Lys-BK. The B 1 R is activated by the active metabolites des-Arg 9 -BK and Lys-des-Arg 9 -BK and has a low level of expression in healthy tissues. The latter receptor is upregulated after exposure to pro-inflammatory cytokines, bacterial endotoxins, and hyperglycaemiainduced oxidative stress [3][4][5][6][7].
Basal expression of B 1 R was reported in the rat and human spinal cord dorsal horn as well as in rat dorsal root ganglion and small caliber primary sensory neurons [30][31][32]. Autoradiographic B 1 R binding sites are increased and distributed all over the grey matter of the spinal cord after peripheral nerve injury [22] and in models of diabetes [7,29,33]. This spatial distribution of B 1 R binding sites suggests that this receptor is not limited to primary sensory afferents but could also be present on spinal cord microglia and astrocytes.
To consolidate the role of B 1 R in pain polyneuropathy, its cellular distribution was investigated in the spinal cord of STZ-induced B 1 R with a newly developed fluorescent agonist named [Nα-Bodipy]-des-Arg 9 -BK (BdABK). The B 1 R selectivity of BdABK was determined by assessing its ability to displace B 1 R ([ 125 I]-HPP-desArg 10 -Hoe 140) and B 2 R ([ 125 I]-HPP-Hoe 140) radioligands by autoradiography. Moreover, the displacement of BdABK fluorescent labeling by B 1 R antagonists (R-715 and SSR240612) was assessed by confocal microscopy. We also investigated the in vivo activity of BdABK in comparison with its native agonist on thermal hyperalgesia in both STZ-treated and control rats. Appropriate selective antibodies were used in confocal microscopy to co-localize B 1 R on astrocytes, microglia and sensory C fibers in STZ-diabetic rats. The induction and overexpression of B 1 R in the spinal cord of STZ-diabetic rats was confirmed by qPCR and autoradiography. Experiments were achieved 4 days after STZ admin-istration because previous studies showed that spinal cord B 1 R was maximally up-regulated and engaged in thermal hyperalgesia 2 days after STZ treatment [9,33].

Animals and treatments
All research procedures and the care of the animals were in compliance with the guiding principles for animal experimentation as enunciated by the Canadian Council on Animal Care and were approved by the Animal Care Committee of our University. Male Sprague-Dawley rats (200-225 g, Charles River, St-Constant, Que., Canada) were housed two per cage, under controlled conditions of temperature (23°C) and humidity (50%), on a 12 h lightdark cycle and allowed free access to normal chow diet (Charles River Rodent) and tap water.

STZ treatment
Rats were used 5 days after their arrival and injected under low light with freshly prepared STZ (65 mg/kg; i.p.; Sigma-Aldrich, Oakville, ON, Canada). Age-matched controls were injected with vehicle (sterile saline 0.9%, pH. 7.0) [33]. Glucose concentrations were measured, with a commercial blood glucose-monitoring kit (Accusoft; Roche Diagnostics, Laval, Que., Canada), in blood samples obtained from the tail vein, in non-fasting animals, before STZ injection, and 4 days after treatment. Only STZ-treated rats whose blood glucose concentration was higher than 20 mM were considered as diabetic.

Tissue preparation for autoradiography and microscopy
Four days after injection of STZ, rats were anaesthetized with CO 2 inhalation and then decapitated. Upper thoracic spinal cord (T3-T7) was removed and frozen in 2-methylbutane (cooled at -40°C following exposure to liquid nitrogen) and stored at -80°C. Few days later, spinal cords were mounted in a gelatin block and serially cut into 20μm thick coronal sections with a cryostat. Thus the sec-tions were thaw-mounted on 0.2% gelatin-0.033% chromium potassium sulfate-coated slides and kept at -80°C for 1 month to allow the adhesion of sections to the coverslip glasses.

Confocal microscopy Slides preparation
On the day of experiments, sections were thawed at room temperature for 10 min to enhance sections adhesion. They were pre-incubated for 10 min in 25 mM PIPES-NH 4 OH buffer (pH 7.4) to allow degradation of endogenous kinins which could occupy receptors. Sections were exposed for 90 min to 50 μM BdABK. Thereafter, slides were washed twice (1 min) in PIPES and fixed with 4% para-formaldehyde [36]. Slides were washed three times (5 min) and then exposed to 1 M of glycine for 90 min to eliminate autofluorescence from aldehyde-fixed tissue. Tissues were permeabilized for 45 min with 0.1% Triton X-100.

Coverslip and microscopy
Slides were washed 3 times (5 min), mounted with coverslip, fixed with mowiol (12 h at room temperature) and stored at -4°C for 1 month or used in confocal microscopy.

SYBR green-based quantitative RT-PCR
Four days after injection of STZ, rats were anaesthetized with CO 2 inhalation and then decapitated. The thoracic spinal cord (T1-T2) was isolated and approximately 10 mg of tissue were put in RNA later stabilization reagent (QIAGEN, Valencia, CA, USA). Total RNA was extracted from tissue according to the manufacturer's instructions. First-strand cDNA synthesized from 400 ng total RNA with random hexamer primers was used as template for each reaction with the QuantiTect Rev Transcription Kit (QIAGEN). SYBR Green-based real-time quantitative PCR using Mx3000p device for signal detection (Stratagene, La Jolla, CA, USA) was performed as described [43]. PCR was performed in SYBR Green Master mix (QIAGEN) with 300 nM of each primer. For standardization and quantification, rat 18S was amplified simultaneously. The primer pairs were designed by Vector NTI software and used [6] ( Table 1).
PCR conditions were as follows: 95°C for 15 min, followed by 46 cycles at 94°C for 15 s, 60°C for 30 s and 72°C for 30 s. The cycle threshold (Ct) value represents the cycle number at which a fluorescent signal rises statistically above background [44]. The relative quantification of gene expression was analyzed by the 2 -ΔΔCt method [45].

Specificity of BdABK
To assess the specificity of BdABK for B 1  BdABK. Each concentration of each competitor was tested on 4 sections per rat from 7 different rats. Those sections were exposed to the film, and total binding was calculated as described above. Moreover, the specificity of BdABK was determined in confocal microscopy by the displacement of fluorescent labeling with the addition of 10 -5 M R- hydrochloride] (kindly provided by Dr Pierre Carayon, Sanofi-Aventis, Montpellier, France) [18] to the incubation medium.

Microglial cell culture
Primary cell culture method Mixed glial cultures were prepared following the protocol of McCarthy and de Vellis [49] with some modifications. Briefly, forebrains were dissected out from one litter of 2-day-old Sprague-Dawley rat pups and the meninges were stripped off before enzymatic and mechanical dissociation. For enzymatic dissociation, HBSS containing 0.25% trypsin (Gibco 15090-046) was used. The tissue-trypsin suspension was incubated for 20 min at 37°C in a water bath with intermittent shaking. After the waiting time for the trypsin digestion is over we added to the tissue-trypsin suspension a mixture of prewarmed DMEM/Dnase I (Sigma DN-25, Dnase I final concentration 0. 25 mg/ml) followed by an incubation for 4 min at 37°C. The resulting suspension was dispersed by a mild mechanical trituration which consisted in the passage through 18-, 22and 25-gauge needles. This cell suspension was then filtred through 70 μm strainer (BD Falcon 352350). After extensive washs in prewarmed HBSS, these dissociated cells were resuspended and plated in 75-cm 2 Falcon tissue-culture flasks (BD Biosciences) previously coated with 10 μg/ml poly-D-lysine (PDL). These mixed cells were growing at 37°C and 5% CO 2 in DMEM (Gibco) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 mg/ml). The media was changed every 2 or 3 days thereafter.
At 10 days-in-vitro, a confluent monolayer of astrocytes was apparent, on top of which oligodendrocyte precursor cells and a loosely attached layer of phase-bright microglia was obtained. Microglias were collected by shaking the flasks for 1 h at 200 rpm at 37°C and 5% CO 2 . Dislodged cells were resuspended and grown in culture medium for microglia [RPMI medium 1640 (Gibco) supplemented with 10% FBS, L-glutamine (1 mM), sodium pyruvate (1 mM), penicillin (100 units/ml), and streptomycin (100 mg/ml)]. The cells were allowed to adhere to the surface of PDL-coated coverslips (30 min at 37°C and 5% CO 2 ), and nonadherent cells were rinsed off.

Microglia cells preparation for confocal microscopy
Briefly, confluent cells were exposed to 300 nM of BK for 24 h to induce B 1 R [50,51]. Control cells were exposed to vehicle. After incubation with BdABK, cell were washed, then fixed and permeabilized with 100% methanol previously stored at -20°C. The fixed cells were then processed as described for immunostaining.

Thermal hyperalgesia
Thermal hyperalgesia was assessed according to the method described by Hargreaves et al., 1988 [52] with minor modifications. Briefly, rats were placed (unrestrained) within a Plexiglass enclosure on a transparent glass floor and allowed to acclimatize for 20-30 min. An infrared beam that constitutes the noxious heat stimulus (Plantar test, Ugo Basile, Italy) was moved beneath the plantar surface of the hind paw. Thermal nociceptive threshold was defined as the latency (seconds) between the heat stimulus (46°C) onset and the paw withdrawal using a feedback-controlled shut-down unit. A cut-off time of 33 s was used to avoid tissue damage. Each paw was tested three times alternatively at minimum intervals of 3 min between stimulation to avoid sensitization of the hind paw. The rats were trained on several days prior to testing B 1 R agonists. Thereafter, the thermal nociceptive threshold was assessed on 3 consecutive days as follows: day 1: baseline, saline and the first dose of des-Arg 9 -BK and BdABK (22.5 μg/kg); day 2: des-Arg 9 -BK and BdABK (225 μg/kg); day 3: des-Arg 9 -BK and BdABK (2250 μg/kg). Agonists were injected intraperitoneally at 1 h apart. This series of experiments was conducted in 3 control and 3 STZ-diabetic rats because the quantity of BdABK available for in vivo study was restricted. Thermal hyperalgesia was calculated as a percentage of the maximum possible effect (% MPE) according to the following formula: % MPE = (100 × (drug latency minus baseline latency)/(cut-off time minus baseline latency)) [9]. The baseline latency corresponds to the average of the first three measurements.

Statistical analyses
All data were expressed as means ± S.E.M. obtained from n rats. Statistical significance was determined with Student's t-test for unpaired samples or a one-way analysis of variance (ANOVA) followed by post-hoc Dunnett test for multiple comparisons. IC 50 values were calculated by Graph Pad Prism 4.0 (GraphPad software, USA). Only probability (P) values less than 0.05 were considered to be statistically significant.  (Fig. 4).

B 1 R fluorescent labeling and selectivity of BdABK
In comparison, same concentrations of Hoe 140 displaced B 2 R binding sites with IC 50 value of 1.33 ± 0.1 nM.

BdABK mediated in vivo thermal hyperalgesia
The in vivo effect of BdABK on pain behavior was assessed by determining its ability to induce thermal hyperalgesia upon intraperitoneal injection in STZ-treated rats. As  expected, BdABK and des-Arg 9 -BK had no significant effect on the nociceptive threshold in control rats, yet both agonists caused thermal hyperalgesia in STZ-diabetic rats at 0.225 and 2.25 mg/kg. These effects were dosedependent and significant when compared to saline or control (Fig. 5). BdABK was however slightly but significantly less potent than des-Arg 9 -BK to induce hyperalgesia at the highest dose. As exemplified by des-Arg 9 -BK, this response peaked at 15 min post-injection and was reversible after 30 min (Fig. 6).

B 1 R mRNA expression assessed by qPCR
A low basal expression of kinin B 1 R mRNA was detected in the spinal cord of control rats (Fig. 7). This expression was significantly increased (18-fold) in the spinal cord of STZ-diabetic rats.

Density of B 1 R binding sites assessed by quantitative autoradiography
As presented in Figure 8, quantitative in vitro autoradiography showed an increase density of specific B 1 R binding sites throughout the grey matter of the thoracic spinal cord in STZ-treated rats when compared to age-matched control spinal cord. B 1 R binding sites (2.4 fmol/mg protein) in spinal cord of STZ-treated rats were 2.7-fold greater than those measured in control rats (0.9 fmol/mg protein). Figure 9 shows the colocalization of BdABK, TOPRO-3 and anti-IBA-1 in STZ thoracic spinal cord. Data suggest that B 1 R is present on spinal microglial cells in STZ-diabetic rats. Figure 10 shows the colocalization of BdABK, TOPRO-3 and anti-IBA-1 in primary microglial cell culture. B 1 R was induced by a pre-treatment with 300 nM BK. About 95 ± 2% of the primary cell culture showed a positive labeling

B 1 R colocalized in primary cultured microglial cells
[Nα-Bodipy]-des-Arg 9 -BK selectivity for B 1 R was evaluated by quantitative autoradiography [Nα-Bodipy]-des-Arg 9 -BK affinity for B 2 R was evaluated by quantitative autoradiography Figure 4 [Nα-Bodipy]-des-Arg 9 -BK affinity for B 2 R was evaluated by quantitative autoradiography. The ability of intraperitoneally injected [Nα-Bodipy]-des-Arg 9 -BK and its native agonist, des-Arg 9 -BK, to alter the paw withdrawal threshold in STZ-treated and control rats Figure 5 The ability of intraperitoneally injected [Nα-Bodipy]des-Arg 9 -BK and its native agonist, des-Arg 9 -BK, to alter the paw withdrawal threshold in STZ-treated and control rats. Data represent maximal effects and are the average of 3 readings taken at 9, 12 and 15 min postinjection in 3 rats per group. Statistical comparison to control (*) and 2250 μg/kg des-Arg 9 -BK (+) are indicated by * + P < 0.05; ** P < 0.01; *** P < 0.001. with anti-IBA-1 confirming cell purity. Data suggest that B 1 R can be induced in vitro on microglial cells. Figure 11 shows the colocalization of BdABK, anti-TRPV1 and anti-CGRP in the thoracic spinal cord of STZ-treated rats. Data suggest that B 1 R and TRPV1 are co-localized on sensory C fibers in STZ-diabetic rats. Figure 12 shows the colocalization of BdABK and anti-GFAP in the spinal cord of STZ-treated rats. Data suggest that B 1 R is also present on spinal astrocyte cells in STZdiabetic rats.

Discussion
This study is using a newly developed selective and high affinity fluorescent ligand enabling the cellular localization of B 1 R on unfixed tissue. It provides the first evidence that B 1 R is localized on microglial cells, astrocytes and sensory C fibers in the thoracic spinal cord of STZ-diabetic rats. This study also highlights the early upregulation of B 1 R (mRNA and binding sites) in the thoracic spinal cord of hyperglycaemic STZ-treated rats.

Diabetes induces B 1 R expression
STZ-diabetic rats provide an accessible model for studying the expression, the pharmacology and physiopathology of the B 1 R in the central nervous system. Pharmacological data showed that functional B 1 R was expressed in spinal cord of STZ-treated rats; its spinal activation led to sympathetically mediated increases of blood pressure and heart rate [53] and to thermal hyperalgesia [9]. Further autoradiographic and functional evidence for B 1 R induction was demonstrated in the lung [54], spinal cord [33], retina [6,55] and brain [56] of STZ-diabetic rats. However, this is the first report on mRNA expression in thoracic spinal cord of STZ-diabetic rats by qPCR. Hyperglycaemia associated with type 1 diabetes can activate NF-κB [57] which is known to induce B 1 R [2,3,58]. Moreover, oxidative stress associated with diabetes was reported to be involved in the induction of B 1 R [6,7,29,59].

[N α -Bodipy]-des-Arg 9 -BK selectivity for B 1 R
Experiments by autoradiography confirm that BdABK is highly selective for B 1  HPP-desArg 10 -Hoe 140, with an IC 50 of 5.3 ± 0.1 nM in thoracic spinal cord of STZ-treated rats. Results also evidenced that B 2 R binding sites were displaced by the selective antagonist, Hoe 140, with an IC 50 value of 1.3 ± 0.1 nM while B 1 R binding sites were displaced by the natural B 1 R agonist, des-Arg 9 -BK (IC 50 = 19 ± 0.2 nM) and by R-715, a selective B 1 R peptide antagonist (IC 50 = 4.3 ± 0.2 nM). Comparison of IC 50 values suggests that the affinity of the B 1 R agonist is increased by the addition of the Bodipy molecule. The stabilization of the N-terminus part of the peptide may contribute to prevent its degradation.
The reason for using 50 μM BdABK was based on preliminary study. The concentration of fluorescent probe needed to get a consistent labeling was higher than the IC 50 value most likely because BdABK binds to B 1 R noncovalently and can be eliminated during the washout period of tissue sections. Signal amplification with radioactivity is also expected to be greater than that achieved with a fluorescent probe. BdABK showed no labeling in thoracic spinal cord of control rats which is in accordance with the inducible character of the B 1 R and its virtual absence in healthy tissues. The elimination of B 1 R labe-ling with BdABK after co-incubation with R-715 or SSR240612 confirms the specificity of the B 1 R fluorescent ligand.
Interestingly, BdABK maintained its biological activity as B 1 R agonist in vivo. Data obtained on the Hargreaves test revealed that BdABK was only slightly less potent than des-Arg 9 -BK to cause thermal hyperalgesia upon peripheral administration. This is consistent with the transient thermal hyperalgesia previously reported in the tail-flick test after intrathecal injection of des-Arg 9 -BK in rats made diabetics with STZ 24 h earlier [9]. Likewise, Gabra and Sirois [24] showed that intraperitoneal administration of des-Arg 9 -BK (400 μg/kg) in STZ-treated rats significantly reduced the paw withdraw threshold in the hot plate and tail-flick test.

Localization of B 1 R B 1 R on microglial cells
Previous work by Noda and coworkers [50,51] showed that B 1 R can be expressed in cultured rat microglia exposed to STZ-treated thoracic spinal cord was exposed to TOPRO-3, a specific fluorescent dye for DNA (A) Rat microglial primary cultured cells were exposed for 24 h to 300 nM BK to increase B 1 R expression Figure 10 Rat microglial primary cultured cells were exposed for 24 h to 300 nM BK to increase B 1 R expression. Then, they were exposed to TOPRO-3, a specific fluorescent dye for DNA BK. We confirmed this result by using our fluorescent ligand in the same condition, thus providing additional evidence of its ability to bind B 1 R in a pure rat microglia model. BK acting via B 2 receptors induces elevation of intracellular calcium leading to the phosphorylation and activation of NF-κB by protein kinase C [60]. NF-κB upregulates B 1 R upon binding to its nuclear promoter [2].
A recent study has demonstrated that B 1 R is involved in microglial migration toward rat brain lesion sites [61]. The presence of B 1 R on spinal microglial cells is in keeping with a recent study suggesting that activated dorsal horn microglia is a crucial component of STZ-induced tactile allodynia, mediated in part, by extracellular signalregulated protein kinase signaling [62]. Importantly, the development of tactile and cold allodynia in a rat model of insulin-resistance was blocked by the B 1 R antagonist SSR240612 [28] and by two antioxidants (N-acetyl-Lcysteine and alpha-lipoic acid) known to prevent the induction of B 1 R [7,29]. Taken together, these results suggest a critical role for microglial B 1 R in generation of tactile allodynia, a manifestation of pain polyneuropathy. It is possible that microglial B 1 R is also involved in STZinduced thermal hyperalgesia as this response was abolished by B 1 R antagonists [5,24,27] and was absent in B 1 R knockout mice treated with STZ [26].

B 1 R on astrocytes
In addition, the present study provides the first evidence that thoracic spinal cord astrocytes bear the B 1 R in STZdiabetic rats. Astrocyte B 1 R may represent another target for neuropathic or chronic pain. Emerging evidence suggests a critical role for astrocytes in the passage from acute to chronic and neuropathic pain. It seems that intracellular calcium level oscillation in astrocytes could spread through astrocytal network and thereby facilitate the formation of new synapses. These new synapses could establish neuronal contacts for maintaining and spreading pain sensation [63]. Moreover, astrocytes are known to release various inflammatory mediators that promote neuroimmune activation and can sensitize primary afferent sensory neurons contributing to development of neuropathic pain [64].

B 1 R on sensory C fibers
Immunohistochemical data showed the presence of B 1 R in DRG and superficial laminae of spinal cord dorsal horn STZ-treated spinal cord was exposed to anti-CGRP, a selec-tive antibody of sensory C fibers (A) Figure 11 STZ-treated spinal cord was exposed to anti-CGRP, a selective antibody of sensory C fibers (A). TRPV1 was labeled with anti-TRPV1 another marker of sensory C fibers (B STZ-treated spinal cord was exposed to anti-GFAP, a specific antibody against astrocytes (A) and to BdABK (B) Figure 12 STZ-treated spinal cord was exposed to anti-GFAP, a specific antibody against astrocytes (A) and to BdABK (B).  [30][31][32]. Those studies suggested a basal expression of B 1 R in primary sensory C fibers of normal rat. This is consistent with the expression of B 1 R in sensory C fibers of STZtreated rats as revealed by the co-localization of B 1 R, CGRP and TRPV1. Horowitz [65] described the crucial role of small A-delta and C fibers in generation of diabetic polyneuropathy and their sensitivity to hyperglycaemia. Ueda's studies [66,67] support the hypothesis that the generation of neuropathic pain is related to alterations in gene and protein expression in primary sensory neurons which could contribute to demyelination of A-delta fibers through the down-regulation of myelin protein such as MBP, MPZ and PMP22. Demyelinated A-delta fibers sprout and synapse with A-beta fibers resulting in the enhancement of pro-nociceptive neurotransmitter release which generated allodynia. The presence of B 1 R on sensory C fibers is in agreement with an earlier pharmacological study that showed that the stimulation of B 1 R with an agonist in the spinal cord of STZ-diabetic rats provokes thermal hyperalgesia via the release of substance P [9].

Basal B 1 R expression in control rats
Authors failed to observe specific fluorescent labelling for B 1 R in normal rats which is rather consistent with the negligible level of B 1 R mRNA and binding sites. Moreover, intrathecal injection of B 1 R agonists or antagonists failed to cause behavioural, cardiovascular or nociceptive responses in control rats, suggesting that the basal expression of B 1 R is not functional in naïve rats [9,53]. Thus the function of the B 1 R detected by immunohistochemistry in the spinal cord of rodents and human remains elusive. It is feasible that B 1 R in control animals is uncoupled to G protein as demonstrated for other G-protein-coupled receptors [68,69]. Although it is possible that the immunological approach is more sensitive, we have evidence (unpublished data) showing that the commercially available B 1 R antibodies (M-19) from SantaCruz Biotechnologies (Santa Cruz, CA, USA) are not specific for immunohistochemical detection since B 1 R labeling persists in spinal cord isolated from B 1 R knockout mice. The latter B 1 R antibodies remain however suitable for Western blot analysis, suggesting that immunohistochemical studies reported with B 1 R antibodies remain to be validated with the appropriate controls in mutant mice.

Conclusion
[Nα-Bodipy]-des-Arg 9 -BK was found selective for B 1 R with an IC 50 value of 5.3 ± 0.1 nM in the rat spinal cord. Furthermore, BdABK maintains its biological activity as agonist as evidenced by its ability to induce thermal hyperalgesia in STZ-treated rats. This new fluorescent ligand enabled the detection of B 1 R in primary microglial cell culture and on microglial cells, astrocytes and sensory C fibers in the thoracic spinal cord of STZ-diabetic rats. Because all these cells have been implicated in neuro-pathic pain, the induction and up-regulation of the B 1 R on these elements consolidate the idea that kinin B 1 R is an important target for drug development in pain processes.