Perivascular histamine receptors mediate vasosensory reflex responses elicited by thermal nociceptive stimuli in anaesthetised rat models

INTRODUCTION

Chemical mediators of inflammation such as histamine, serotonin, bradykinin, substance P and ATP play a significant role in the pathogenesis of diseases affecting the blood vessels, namely migraine, myocardial infarction, peripheral vascular diseases and atherosclerosis.^[1] The contribution of inflammatory mediators (algogens) present in peripheral blood vessels in the reflex modulation of the cardiorespiratory (CVR) system has been described by several workers.^[2-4] Reflex CVR alterations produced after intra-arterial (i.a) instillation of algogens into a local segment of medium-sized peripheral blood vessels are known as ‘vasosensory reflex responses.’^[2,3] The walls of blood vessels particularly arteries are richly innervated by sensory nerve terminals.^[5] Transient receptor potential (TRP) cation channels particularly the Vanilloid receptor-1 subfamily are located in the perivascular nociceptive terminals.^[6-8] These afferent nerves when activated by nociceptive chemical, thermal and mechanical stimuli convey the information about the noxious damages in the vicinity to nuclei located in the pons and medulla, which modulate the CVR parameters by reflex mechanisms.^[5]

Perivascular nociceptive afferents are known to be activated by a variety of chemical stimuli such as capsaicin, resiniferatoxin, metabolites of polyunsaturated fatty acids and noxious heat.^[9-12] The vasosensory reflex responses evoked by chemical agents such as capsaicin, anandamide, ab Me-ATP and bradykinin have been described elsewhere.^[2,3,13] Works from our laboratory have demonstrated similar reflex responses using histamine, bradykinin and Mesobuthus tamulus (BT) venom as nociceptive tools.^[4,14-18] Although vasosensory reflex responses elicited by chemical nociceptive stimuli have been studied extensively, the same is not true with thermal and mechanical nociceptive stimuli. Therefore, the present study was conducted to study the nature of vasosensory reflex responses elicited by thermal nociceptive stimuli (warm and cold) and to evaluate the role of histamine, a principal mediator of inflammation in mediating these responses. Further, the novel experimental design used in this study facilitated the deposition of nociceptive agent in the local intravascular compartment of femoral artery, thereby stimulating the perivascular afferents with precision.^[18]

MATERIALS AND METHODS

The Animal Ethical Committee of our Institute approved the present study (Ref. No. Dean/2019/IAEC/1627 dated 17.11.2019). All animals were handled in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and EU Directive 2010/63/EU for animal experiments. Chemicals (saline/pheniramine) were instilled into the femoral artery retrogradely through cannula evolved in our laboratory and the volume of injectable was kept minimal (0.1 ml). Six animals were used in each group and the laboratory temperature was maintained at 30°C. At the end of experiments, animals were sacrificed by overdose of anaesthesia. The detailed methodology of dissection, cannulation and recording was standardised, validated and published in our previous articles^[4,18] and a relevant description is given below.

RESULTS

Instillation (i.a) of warm saline produced immediate (2–6 s) hyperventilatory, hypotensive and bradycardiac responses in contrast with equivolume of normal saline at room temperature (30°C). Cold saline produced similar pattern of CVR changes like warm saline but of lesser magnitude, which were not significantly different as compared to the normal saline [Table 1; Figures 1 and 2]. Since warm saline produced optimal responses on all CVR parameters, it was used for the elicitation of vasosensory reflex responses in the subsequent sets of experiments. CVR parameters were recorded for 10 min after instillation of saline but it was found that no significant changes in the CVR parameters took place after 60 s of instillation.

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Figure 1:

Original recordings showing the effect of intra-arterial instillation of cold saline (0°C) and warm saline (55°C) on respiration (Resp), blood pressure (BP) and electrocardiogram (ECG) as compared with the effects of normal saline at room temperature (30°C). The recordings depict 10 s before and 40 s after instillation of saline. The time scale is shown in the lower panel. Dotted lines indicate the point of instillation of saline.

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Figure 2:

Time-response plots showing the effect of intra-arterial instillation cold (0°C) and warm saline (55°C) on respiratory minute volume (RMV), mean arterial pressure (MAP) and heart rate (HR). The control responses after instillation normal saline at room temperature (30°C) are given in each row along with cold saline responses. The values are in mean±SEM from six experiments. An asterisk (*) indicates significant difference in the responses as compared to normal saline control group (P<0.05, post hoc Dunnett’s test). Arrows indicate the point of instillation of saline.

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Latencies of the CVR responses

The duration of onset of responses (latency) was different for different CVR parameters with different temperatures of saline [Figure 3]. Latency was shortest for HR and longest for MAP responses. The latencies for hypotensive responses were 5.83 ± 0.28 s and 5.50 ± 0.55 s for warm and cold saline, respectively. The latencies for hyperventilatory responses were 2.17 ± 0.17 s and 2.17 ± 0.11 s for warm and cold saline, respectively. The latencies for bradycardiac changes were 1.42 ± 0.20 s and 1.25 ± 0.17 s for warm and cold saline, respectively. It was observed that the difference in the latencies of MAP, RMV and HR responses with warm saline was not statistically significant from the cold saline group [Figure 3; P > 0.05, unpaired t-test] but the latencies of MAP responses were significantly different as compared with the latencies of RMV and HR responses [Figure 3; P < 0.05, unpaired t-test]. Normal saline at room temperature did not produce any significant response on the CVR parameters so the latency cannot be calculated for this group.

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Figure 3:

Bar diagrams showing the latencies of mean arterial pressure (MAP), respiratory minute volume (RMV) and heart rate (HR) responses for cold (0°C) and warm saline (55°C). The values are expressed as mean±SEM from six experiments. The latency of MAP responses is significantly different as compared to the RMV and HR responses (P<0.05, unpaired t-test).

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Pheniramine pre-treatment attenuated warm saline-induced CVR changes

Instillation (i.a.) of pheniramine per se did not produce any change in the resting RMV, MAP and HR. However, pheniramine pre-treatment attenuated the warm saline-induced hyperventilatory responses (from 89% to 24% of initial value), hypotensive responses (from 26% to 6% of initial value) and bradycardiac responses (from 24% to 3% of initial value) significantly [Table 1; Figure 4; P < 0.05, post hoc Dunnett’s test].

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Figure 4:

Pheniramine pre-treatment (+Phen) blocked the warm saline (WS)-induced responses. The time-response plots showing the effect of intra-arterial instillation of WS on respiratory minute volume (RMV), mean arterial pressure (MAP) and heart rate (HR) in pheniramine pre-treated (+Phen +WS) group and WS (55°C) only group are shown in (a-c). An asterisk (*) indicates significant difference in the CVR responses in +Phen +WS group as compared to the WS only group (P<0.05, post hoc Dunnett’s test). Arrows indicate point of instillation of saline. (d) The original recordings showing the effect of WS-induced changes on respiration (Resp), blood pressure (BP) and electrocardiogram (ECG) in pheniramine pre-treated animals. Dotted line indicates the point of injection of WS after +Phen.

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DISCUSSION

The present observations demonstrate that instillation of warm saline into a segment of femoral artery elicits immediate hyperventilatory, hypotensive and bradycardiac changes of shorter latency, as compared with equivolume of normal saline at room temperature, excluding the possibilities of stretch/ischemia-induced responses on the arterial wall. Cold saline also produces similar pattern of CVR changes as that of warm saline but of lesser magnitude which is not significantly different from the control group. All the responses are short-lived and CVR parameters return back to the initial level within 30 s after instillation of nociceptive agents. Pheniramine pre-treatment significantly attenuates warm saline-induced reflex CVR alterations.

Studies elsewhere showed that around 45% of nociceptors (TRPV-1) are activated by temperatures of >45°C and around 60% of them are stimulated by temperatures of >52°C.^[19-23] Therefore, in our experiments, the temperature of warm saline was kept at 55°C so that adequate thermal stimulus could be delivered to the perivascular nerve endings, as a dissipation of 1–2°C is expected in the process of instillation. Regarding cold, only around 20% of nociceptors (TRPM-8) are activated by temperatures of <4°C.^[24-28] Hence, the temperature of cold saline in our experiments was chosen at 0°C. Utmost care was taken at every step to minimise the temperature dissipation, by ensuring that the saline is instilled into femoral artery within 2 s of measurement of its temperature. Further, temporary alterations in the properties of receptor proteins are expected after exposure to higher temperatures while such alterations are known to be minimum with colder temperatures.^[29,30] Hence, very high temperatures (>60°C) were avoided in this study as it may permanently denature the perivascular receptors.

Workers in the past have shown CVR alterations elicited by intravascular injection of various chemical nociceptive agents such as capsaicin, ATP, anandamide, cinnamaldehyde, acrolein, histamine and bradykinin.^{[2-4,13,18,31,32]} Earlier works from our laboratory have also shown the reflex responses elicited by i.a instillation of Indian red scorpion venom as nociceptive agonist.^[14-17] In the present study, the vasosensory reflex responses are elicited by activating perivascular afferent neurons using thermal nociceptive stimuli. Further, in this study, a 24G double ported cannulation of femoral artery is performed to instil the nociceptive agonist and to record the BP at the same time.^[18] This minimises the distress to the animal by eliminating the need for separate carotid artery cannulation for BP recording as performed earlier. The cannula also facilitates the deposition of the nociceptive agents in a local segment of femoral artery, thereby eliciting vasosensory reflex responses more precisely.

The shorter latencies of responses show that the CVR changes observed in the present study are reflex mediated responses as humoral responses cannot exhibit so quickly. The perivascular nociceptive afferents contain different nerve fibre types, namely fast Aq and slow C fibres. The different latencies of RMV, MAP and HR parameters support the above ideology and theoretically suggest the possible existence of different sets of afferents of different nerve fibre types, for different CVR parameters. In this study, it can be observed that MV, MAP and HR responses elicited by warm saline were significantly attenuated after pheniramine pre-treatment. Histamine receptors (H₁ and H₂) are already known to be located on the vascular endothelium. Works elsewhere have demonstrated that activation of histamine receptors potentiates and even cross-activates vascular TRP receptors and vice versa.^{[11,25,33-35]} This cross-activation between perivascular receptors is a major contributor to the nociceptive responses elicited by chemical mediators of inflammation in health and diseases. The attenuation of warm saline-induced responses by pheniramine in our experiments points out the role of perivascular histamine receptors in mediating vasosensory reflex responses elicited by thermal nociceptive stimuli. However, interaction of receptors at the molecular level and pathways responsible for the reflex CVR changes needs to be explored further.

CONCLUSION

Thermal stimuli of appropriate strengths elicit vasosensory reflex responses altering CVR parameters that is, RMV, MAP and HR. Pheniramine, a non-selective histamine receptor antagonist, attenuates these responses indicating the role of perivascular histamine receptors in producing the temperature-induced responses. The activation of perivascular nociceptors by thermal stimuli demonstrates the modulation of CVR parameters due to the local changes in the blood temperatures within peripheral vascular compartments (e.g. blood vessels adjacent to the exercising muscles) in contrast to the systemic temperature changes as occurring in fever and hyperthermia. Our study thus supports the hypothesis regarding the involvement of medium-sized peripheral blood vessels in the reflex modulation of the CVR system in states of health and diseases.