age related pain sensation change
TRANSCRIPT
Age-‐related changes in the primary afferent function in vitro
Liang Huang, Ratan Banik
New Jersey Neuroscience Institute
Abstract
The altered pain perception and the cutaneous nociception elicited by noxious stimuli to the skin during senescence are not well understood, and it is thought that this could in part be due to changes in peripheral pain sensing processes. We systematically examined cutaneous nociceptor responses and nociceptive behaviors in young (2-‐6 months) and in aged (18-‐26 months) F334/N rats. C-‐fiber nociceptors in the skin were identified by mechanical stimulation, and extracellularly recorded from hind paw skin-‐saphenous nerve preparations in vitro. The aim of the present study was to investigate the activities of aged skin nociceptors systematically to mechanical, chemical stimuli, and to compare with the data from young animals. Mechanical threshold measured by a ramp mechanical stimulus in the aged skin was significantly higher than that in the younger skin. The latency to chemical stimulations tended to be longer. In addition, the magnitude of the chemical response during the 60s chemical stimulus was not significantly different. In contrast, the numbers of total net discharges induced by chemical (bradykinin, prostaglandin, serotonin, histamine) stimuli were not different with the different ages. After sensitization by chemicals, the young rats displayed a stronger and longer mechanosensitization. This showed for the first time that not only receptive properties of afferent terminals but also mechanical sensitizations by chemicals in axons are changed in aged rats. These results showed decreased mechanical and chemical responses in skin C-‐afferents in the aged rats.
Introduction
With advancing age, a decline in the sensation is well reported to occur. Ageing influences on morphological and functional features of cutaneous mechanical transducers and mechanosensitive ion channels, sensory innervation, neurotransmitters and even vascular system required to ensure efferent function of the afferent nerve fibres in the skin. This, in conjunction with effect of ageing on the skin per se and central nervous system, could significantly affect the skin sensation among the ageing population. However, little is known about the peripheral neural mechanisms of skin nociception in the aged.
Ageing is associated with reductions of the principal functions of the skin, including protection, excretion, secretion, absorption, thermoregulation, pigmentogenesis, and regulation of immunological processes and wound repair. Ageing is also associated with a progressive decline in cutaneous thermal, vibratory and mechanical sensory perception (Guergova S., Thermal sensitivity in the elderly: a review Ageing Res. Rev., 10 (2011), Lin Y.H., Influence of aging on thermal and vibratory thresholds of quantitative sensory testing. J. Peripher. Nerv. Syst., 2005 and Taguchi, 2010). However, the change with age in pain perception in humans and the nociceptive behaviors in animals elicited by noxious stimuli to the skin are not well understood, and little is known about the peripheral neural mechanisms of cutaneous nociception in the aged and responses to mechanical stimulation and to inflammatory soup
were not recorded. The sensitizations of mechanical response by inflammatory soup from different age groups remain unclear. To date, nearly all attempts to characterize aged afferent fibers have utilized structural, biochemical, or molecular measures1-‐12. Morphologic studies reported several abnormalities after aging such as demyelination, axonal atrophy, reduction in the expression of cytoskeletal proteins5,9,10. Biochemical studies found reduction of neuropeptide expression13 and molecular studies found reduction in the expression of the molecules necessary for transduction of natural stimuli6,7,11. Using an in vitro skin-‐saphenous nerve preparation, single-‐fiber recordings were made from mechano-‐heat sensitive C-‐fiber nociceptors innervating rat glabrous hind paw skin, and their responses were compared with those obtained from different age groups. Responses to mechanical stimulation and to inflammatory soup were tested. The sensitizations of mechanical response by inflammatory soup from different age groups were also investigated.
Methods
Animals
Experiments were performed on 52 male F344/N rats of various ages. Two months (n=13), 6 months (n=22), 18 months (n=6), and 26 month (n=11) old rats were purchased from National Institute of Aging, Bethesda, Maryland, USA. Two to four animals were placed in plastic cages with sawdust bedding and housed in a climate-‐controlled room under a 14/10 hr light/dark cycle. The Animal Care and Use Committee at The Seton Hall University, South Orange, New Jersey, USA has approved experiments, and the animals were treated in accordance with the Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals.
Organ bath
Electrophysiological recordings were performed in animals. Animals were killed using CO2 inhalation; then hairy skin of the rat hind paw and its intact saphenous nerve were dissected free from muscles and tendons. The preparation was then placed in an organ bath and was continuously super fused with a modified Krebs-‐Henseleit solution (in mM: 110.9 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2So4, 24.4 NaHCO3, and 20 glucose), which was saturated with a gas mixture of 95% O2 and 5% CO2. The temperature of the bath solution was maintained at 34 ± 1°C. After dissection, the preparation was placed with ‘epidermal side down’. The nerves attached to the skin were drawn through one small hole to the second chamber, which was filled with liquid paraffin. The nerves were placed on a fixed mirror, their sheaths removed and nerve filaments repeatedly teased to allow single fiber recording to be made by using double-‐platinum electrodes (one for recording and another for reference). Single nociceptive afferent fibers were recorded extracellularly with a differential amplifier (DAM50, Harvard Apparatus, Holliston, MA). Neural activity was amplified and filtered using standard techniques. Amplified signals were led to a digital oscilloscope and an audio monitor and fed into PC computer via a data acquisition system (spike2/CED1401 program). Action potentials collected on a computer were analyzed off-‐line with a template matching function of spike 2 software.
Identification of afferents
The search strategy was mechanical stimulation by a fire-‐polished glass rod; thus, mechanosensitive afferents were characterized. Only units with a clearly distinguished signal to noise ratio were further studied. Rapidly adapting, low threshold A-‐β and D-‐hair fibers were not studied. After the initial assessment, fibers were evaluated for their responsiveness to controlled mechanical stimuli and a cocktail of chemicals, previously termed as ‘inflammatory soup’. In this study, ingredients of this soup and concentrations were different from other studies and the pH was normal (7.4). Aliquots (20 µl) of chemical cocktail were prepared by combining bradykinin, serotonin, and histamine dissolved in distilled water with prostaglandin E2 dissolved in dimethyl sulfoxide (DMSO) and stored at -‐20°C. The aliquots were diluted to final concentration (10 µM) in neutral (7.4) Krebs’ solution on the day of the experiment. All chemicals were obtained from Sigma-‐Aldrich (St. Louis, MO). The decision to use 10 µM concentration of the soup was based on the results of published studies (14,15).
Conduction Velocity and Fiber Categorization
In this study we concentrated on the C-fiber nociceptors. The conduction velocity was always measured at the end of the experiment to avoid damage to the receptive field or alteration of fiber properties. The conduction velocity of the axon was determined by monopolar electrical stimulation through an epoxy-‐coated electrode. The electrical stimulation (1-‐20 V at 0.2-‐1 Hz for 0.5-‐2 ms) was delivered at the sensitive spot of a receptive field. The intensity of the stimulus started form 0.1 V and gradually increased until the similar shape spike appeared. The distance between receptive field and the recording electrode (conduction distance) was divided by the latency of the action potential (stimulus artifact to the appearance to spike).
The fibers were classified using criteria from Leem et al.16 Afferent fibers conducting slower than 2.5 m/s were classified as C-‐fibers, those conducting between 2.5 m/s and 24 m/s as Aδ-‐fibers, and those conducting faster than 24 m/s as Aβ-‐fibers. Units were classified as mechanosensitive nociceptors on the basis of their graded response throughout the innocuous and noxious range of mechanical force stimuli. Rapidly adapting fibers were not studied.
Feedback –controlled mechanical stimulation
To measure quantitative mechanosensitivity, a servo force-‐controlled mechanical stimulator(Series 300B Dual Mode Servo System; Aurora Scientific, Aurora, Ontario, Canada)17were used. A flat and cylindrical metal probe (tip diameter, 0.7 mm) attached to the tip of the stimulator arm was placed just close to the receptive field so that no force was generated. Servo-‐controlled mechanical stimulation (Series 300B dual mode servo system, Aurora Scientific, Canada) was used to measure mechanosensitivity. The computer controlled ascending series of square-‐shaped force stimuli was applied to the most sensitive spot of the receptive field at 60-‐s intervals. Since the neural responses of cutaneous mechanosensitive nociceptors to mechanical stimuli are highly correlated with compressive stress (force) than compressive strain ( displacement),17 sustained force-‐controlled stimuli ( rise time, 100ms; duration of sustained force plateau, 1.9s) were applied. Each force stimulus was 2s in duration and started from zero to 12, 32, 52, 72, 92,112,132,151,171,191,210mN. When an afferent produced a response to a particular force controlled ramp, it received additional ascending series of stimuli to construct stimulus response curve (as shown in Fig. 1). The total number of spikes generated during ascending series of force pulses before
112mN is compared between different age groups (see Fig. 1). The mechanical threshold of units was determined when an afferent produced a response to a particular force controlled ramp.
Chemical Stimulation
After mechanical stimulation, chemosensitivity was assessed using modified Krebs-‐Henseleit solution. To restrict the chemical stimuli to the isolated receptive field, a small metal ring ( internal diameter, 5 mm; height, 6 mm; volume, 0.4 ml), which could seal by its own weight, was used. In some cases, inert silicone grease was added to ensure a waterproof seal.
After recording baseline for 5 min, the metal ring was placed and the Krebs-‐Henseleit solution inside the ring chamber was removed and a chemical cocktail, commonly present in an inflammatory milieu, was applied to the receptive field for 60 s with a temperature of 32 °C. The RF was continuously superfused with Krebs solution (32 °C) before and after application of chemical soup.
We compared the latencies for a response which was calculated with time from onset of chemical application to appearance of two or more consecutive discharges exceeding the mean frequency + 2 SD of the background discharge rate during the control period (60 s). We also compared the mean frequency or total spikes during a response between different groups (see Fig. 2).
Following chemicals were used to prepare this chemical cocktail: bradykinin, histamine, serotonin, and prostaglandin E2. The pH of this cocktail was normal (7.4). The concentrations are 10-‐6M, these concentrations were determined according to past literature (Kessler W 1992) and our pilot study. Ten min after chemical application, computer controlled ascending series of square-‐shaped force stimuli was applied in a few experiments. These data were used to compare changes in the mechanical stimulus response before and after chemical soup (see Fig. 3).
Response criteria for chemical stimulations
When a fiber fulfilled the following criteria, it was defined to be sensitive to a stimulus: (1) the net increase in the discharge rate during the application period of 60 s for chemical soup was more than 0.1 imp/s from the background discharge rate during the control period (60 s) immediately before application, and (2) the instantaneous discharge rate of two consecutive discharges exceeded the mean + 2 SD of the background discharge rate.
Data analysis
Action potentials collected on a computer were analyzed off-‐line with a template matching function of spike 2 software. Quantitative analysis was carried out by counting total impulses generated in the stimulation period. In addition, average discharge frequencies during chemical soup application were also counted. Only good signal-‐to-‐noise ratio (>2:1) was considered.
Statistical analysis
Results are expressed as median with interquartile range (IQR). Averaged response patterns of afferents are shown with mean ± SEM. Comparisons of the electrophysiological data between the young and the aged rats were done using the Mann–Whitney U-‐test. Mann–Whitney U-‐test was also used to compare baseline (before application of inflammatory soup) spike numbers induced by mechanical stimulation and the spike numbers after inflammatory soup. All tests were made with GraphPad Prism software, version 5 (GraphPad, San Diego, CA). Values of p <0.05 were considered significant.
Results
1 General properties of C-‐fibers from young and senescent animals
127 fibers were identified. 86 C-‐fiber nociceptors innervating the hairy skin of rat hindpaw were studied: 64 from the young rats and 22 from aged rats. Conduction velocity was not different between two age groups. The conduction velocities of the control C-‐fibers ranged from 0.1 to 1.5 m/s (0.54 ± 0.06 m/s, IQR: 0.1-‐1.0m/s), and those of the aged C-‐fibers were between 0.1 and 1.2 m/s (0.59 ± 0.09 m/s IQR:0.18-‐1.2m/s). Part of the reason we found so few mechanically sensitive and chemically sensitive C-‐fibers in aged rats when compared with young rats might have been a reported remarkably decreased proportion of mechano-‐responsive C-‐fibers and an notable increase in the proportion of mechano-‐insensitive C-‐fibers in aged rats (Taguchi 2010), which phenomenon is also found in humans(Namer, 2009). Since we only identify the C-‐fibers by using manual probing with a glass rod, therefore, we found much less mechano-‐responsive c-‐fibers in aged group in comparison with young group.
There was no significant increase in the discharge rates of spontaneous activity, which were 0.05 imp/s (IQR: 0-‐0.14 imp/s) in young rats and 0.02 imp/s (IQR: 0-‐0.10 imp/s) in old rats, respectively. In this study all tested C-‐fibers responded to the inflammatory soup stimulation, and they had a single spot like receptive field.
2 The mechanical thresholds are different between youth and aged rats.
Although the Primary afferent response to mechanical stimuli of different age groups looks the same, the mechanical thresholds are different between youth and aged rats. Mechanical threshold measured by a train mechanical stimulus in the aged skin median; 68.44 mN (IQR: 52.1–92.1 mN), n = 18) was significantly higher than that in the younger skin (median; 52.67 mN (IQR: 33.6–72.0 mN), n = 57, p < 0.05, Mann–Whitney U-‐test). In addition, the magnitude of the mechanical response during the first 6 stimulus (from 13mN to 112mN) was significantly lower in the aged skin (22.5 spikes (IQR: 10.75–34.25 spikes)) than in the young (31.0spikes (IQR: 24.25–42 spikes), p < 0.05, Mann–Whitney U-‐test).
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Figure 1 Primary afferent responses to mechanical stimuli. (a–g) Digitized oscilloscope tracings of afferent responsive to mechanical stimuli. Single action potentials were recorded from fine filaments teased from medial or lateral plantar nerves of control mice while the receptive field was stimulated by a feedback-‐controlled force stimulator. Seven consecutive recordings show increasing responses to the ascending series of force (h) stimuli. The stimulus duration of each pulse was 2s and they were delivered at 30 s intervals. (i) Comparison of mechanical response thresholds between <6 months old (n=57), >18 months (n=18) rats.
2 Senescent rats have longer latency in responses to inflammatory soup.
The onset of neuronal response to chemical stimulation was significantly delayed in the afferents from senescent rats. In the aged mechano-‐responsive C-‐nociceptors the response latency to inflammatory soup (median: 15 seconds, (IQR: 9-‐23 seconds)) was significantly longer than that in the younger skin (median: 10 seconds, (IQR: 8-‐14 seconds)) (p < 0.05, Mann–Whitney U-‐test), while the magnitude of the response was not different between the two age groups. Intensity measured by total net spikes in the aged skin (median: 210.5 (IQR: 157.3–281.5), n = 20) was no different from that in the young skins (median: 194.0 (IQR: 139.3-‐319.8), n = 44, p =0.93, Mann–Whitney U-‐test).
Our observation suggests that initiation of chemosensitivity within afferents from senescent rats is slow but once they are activated they can produce a same response as afferents from younger rats, which were proved by the same numbers of total net spikes induced by inflammatory soup in young and aged rats.
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Figure 2 Specimen records from 4 single primary afferents 2 months (a), 6 months (b), 18 months (C) and 26 months (d) rat hairy skin during trials of inflammatory (mixture of chemicals present in an inflammatory condition). Ordinate: frequency of discharges; ‘inflammatory soup’ was applied for 1 min. Comparison of latencies(e), which was calculated with time from onset of chemical application to appearance of clear response. (f) Comparison of mean frequency (Mann-‐Whitney test) (g) total spikes during a response.
3 Inflammatory soups sensitize mechanical responses of both young and senescent rats. However young rats show longer and stronger sensitization.
Application of inflammatory soup had sensitization effect on mechanical responses. Before and after inflammatory soup application, a series of mechanical stimulation were applied to get mechanical response curve. Inflammatory soup was super perfused for 1 min. The mechanical stimulus response curves before and after inflammatory soup application for young rats (n=34), and aged rats (n=12) are respectively shown in a,b. Ordinate: total spikes/stimulation.
We compared the spikes number between the baseline mechanical responses of nerve fibers and that of after inflammatory soup application of the same fiber. All numbers were counted at the same force level on the same fiber before and after chemical soup application. Therefore, even after inflammatory soup application, the same fiber might fire at different threshold force, mostly at a lower threshold; we still counted the number of spikes that occurred at the same force level as the thresholds and stimulation intensities indicated by baseline mechanical responses.
In the hairy skin preparation, application of the chemical soup caused the afferent firing rate to be significantly increased during controlled mechanical stimuli. (One sample t-‐test). The soup enhanced firing rate in young rats during controlled mechanical stimuli. The percentages of spikes compared to baseline in each phases are: s1: 163.4% s2:225%; s3: 199%; s4:190%, s5:181%; s6:141%, which are significantly higher from s2 to s5 compared to baseline (Fig. 3). In aged rats, the percentages are: 91%, 88%, 132%, 184%, 161%, and 128% respectively. In aged skin, during the s1, s2, s3, s5 and s6 phase of controlled stimulation after soup, there was no significant change in activity (Fig. 3). Compared to young animals, the sensitizing effect of the chemical soup on the old animals was only seen significant at S4 after soup administration. Also, the percentages of changes in firing rates are different. Compare to aged skin, the extent of increases in s1 to s2 in young skin were higher (s1 P=0.047, s2 P=0.037) (Fig 3 c). A specimen recording showing the excitatory effect of soup on afferent nerve activity during controlled stimulation of a young and old rat can be seen in Fig. 3. Figure 3
Specimen demonstrating the sensitizing effect of sp during normal and stimulation is shown in fig a,b. Application of inflammatory soup had sensitization effect on mechanical responses. Before and after inflammatory soup application, a series of mechanical stimulation were applied to get mechanical response curve. Inflammatory soup was super perfused for 1 min. The mechanical stimulus response curves before and after inflammatory soup application for young rats (n=34), and aged rats (n=12) are respectively shown in a,b,c. Ordinate: total spikes/stimulation.
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Fig 3 Effect of inflammatory soup (10-‐8M) on skin afferent activity in young (black squares) and aged (open triangles) rats. Neural activity is shown in response to mechanical stimulation of the skin compared to pre-‐soup control (represented by solid line set at 0%). The sensitizing effect of
inflammatory chemicals was seen in s1-‐s6 in young skin, although it is only seen in s4 in aged skin. Data are shown as mean ± SEM. *P < 0.05; one sample t-‐test; n=34 fibers for young rats; n=12 fibers for aged rats. Discussion
Ageing is of interest because ageing influences morphological and functional features of cutaneous mechanical transducers and mechanosensitive ion channels, sensory innervation, neurotransmitters and even vascular system in the skin(Ageing Res Rev. 2014 Jan;13C:90-‐99.Effect of ageing on tactile transduction processes. Decorps J.). Some age-‐related disappearances in epidermal C-‐fiber endings were previously reported to be earlier or more markedly than those in myelinated fiber endings (Pare et al., 2007 and Ceballos et al., 1999). The response to chemical is of interest because ageing might have notable effect on different response to endogenous or exogenous substance such as bradykinin, histamine, and prostaglandin. In addition, there could be different sensitization process in aged compared with young animals.
The present study assessed in rats whether there is an ageing related pain sensation change. We found that although the net intensity has no difference between young and senescent rats, aged rats developed a relative longer latency in response to chemical stimulus. In addition, young rats showed lower mechanical threshold and stronger mechanical response to stimulation. Also young rats presented a stronger sensitization of mechanical response after chemical stimulation compared to senescent rats.
It is generally agreed that the cool and warm detection thresholds assess the function of small myelinated Aδ fibres and unmyelinated C fibres, whereas sensitivities to vibration and tactile stimulation assess the function of large myelinated fibres, respectively (Campero et al., 1996 and Verdugo and Ochoa, 1992). Abnormalities of the sensory system, such as detection thresholds, nerve conduction velocities, structural changes of sensory fibres can also develop because of ageing. For example, modest functional abnormality of small sensory fibres was shown in the older subjects, who displayed increased warm detection threshold compared to young adults (Fromy et al., 2010). Also the degree of activity-‐dependent conduction velocity slowing in response to high frequency stimulation was more pronounced in aged subjects (Namer, 2009). These changes in the axonal properties of C-‐fibres in aged subjects are compatible with hypoexcitability of the fibers.
Decreased mechanical response
It was suggested that the ratio of mechano-‐responsive fibres to mechano-‐insensitive fibres was shifted in favor of the mechano-‐insensitive fibres in older subjects (Namer et al., 2009 and Orstavik et al., 2006, Taguchi 2010 pain). However, since we used the probe stimulation to identify only mechano-‐responsive C-‐fibers instead of electrically identifying both mechano-‐responsive and mechano-‐insensitive C-‐fiber population, we did not see such a ratio shifting. But we found much less mechano-‐responsive fibers (22) in our aged group compared with young rats (64) which might partially be explained by the ratio shifting from mechano-‐responsive dominant fibres to mechano-‐insensitive fibres.
Our results showed a higher mechanical threshold of response in the aged group in comparison to young rats, which is well in line with the previous observation in SD rats (Taguchi 2010 pain). The mechanical response of individual mechano-‐responsive c fibres tends to decrease with age. This may resulted from following reasons: First, the ageing effects on the structure and function of these mechanosensitive ion channels could contribute to the age-‐related mechano-‐ response. Activation of mechanosensitive ion channels is important for the detection of mechanical stimuli required for transduction to electrical signals in sensory neurons. Expression of sodium channel Nav1.8 and TRPV1 expression has been shown to be lowered in cutaneous nerves of aged mice (Wang s, neurobiol Aging, 2006) and is related to reduced thermal sensitivity. The GFRalpha3 receptor, which binds the growth factor artemin and is expressed by TRPV1-‐positive neurons, was also decreased in the DRG of aged animals. These findings indicate that loss of thermal sensitivity in aging animals may result from a decreased level of TRPV1 and Nav1.8 and decreased trophic support that inhibits efficient transport of channel proteins to peripheral afferents. Beside, some findings have shown that selective TRPV1 antagonists cause a reduction in both thermal and mechanical hyperalgesia and TRPV1 also plays a role in mechanical hyperalgesia (Pomonis et al., 2003; Walker et al., 2003; Tang et al., 2007; Btesh J, 2013).
ASIC 3channel has also been shown to detect some cutaneous touch and painful stimuli (Fromy, 2012). Other ion channels such as TRPA1, MEC4/MEC-‐10 and two-‐pore domain potassium (K+)-‐selective channels (such as TREK1 and TRAAK) might also be playing as a neuronal mechanosensitive channel (Decorps J, 2014). Although the ageing effects on the structure and/or the function of these mechanosensitive ion channels are not described, one can speculate that they could contribute to the age-‐related tactile defect.
Second, changes in the physical properties of aged skin may influence the nociceptor response. There are pronounced age-‐induced changes in the viscoelastic properties of the skin and underlying tissue. Profound differences in some mechanical properties of the skin were found between young and adult rats. The compliance of the skin is decreased in adult rats when compared with young rats18(Baumann KI, Hamann W, Leung MS: Mechanical properties of skin and responsiveness of slowly adapting type I mechanoreceptors in rats at different ages. J Physiol 1986; 371: 329-‐37)
During rats’ adulthood, there was a subsequent tortuosity of the distorted elastic fibers which have lost their original elasticity and interlock with the collagen bundles. Interlocking of both collagen and elastic fibers decrease tissue compliance19(Imayama S. Am J Pathol 1989). In human being, the thickness of the dermis also decreases with age and this is accompanied by a decrease in number of mast cells and fibroblasts, and a decrease in the generation of collagen, elastin, glycosaminoglycans, and hyaluronic acid. It is thought that changes in the amount of collagen, alterations in tissue reactive oxygen species or decreases in the amount of fibroblast-‐collagen linkage may result in a diminished ability of the skin to detect or propagate mechanical stimuli; however, it has not yet been investigated. 20 (Wu M: Effect of aging on cellular mechanotransduction. Ageing Res Rev 2011).
We also found that in aged rats, the number of impulses (magnitude of response) induced by mechanical stimulation tend to decrease compared to young rats, which could be due to the following reasons: First, since there are decreased expression of Nav1.8 and TRPV1 protein in cutaneous nerves
of aged mice (Wang s, neurobiol Aging, 2006). It has been indicated that Nav1.8 sodium channels contribute substantially to action potential electrogenesis in DRG neurons (J Neurophysiol. 2001, Renganathan). It is possible that the age-‐related expression of Nav1.8 could lead to changes in less action potential electrogenesis in aged rats. Secondly, a decreased sodium-‐ potassium pump activity in dorsal root in aged mice was observed (Robertson, 1993). As it has been suggested this decreased basal level of pump activity would lead to relatively depolarized membrane potential and higher proportion of inactivated sodium channels, which would result in hypoexcitability of fires to sensory stimuli (Namer 2009). This could also leads to fewer spikes to mechanical stimulation in aged skin.
Chemical responses and sensitized mechanical response after chemical soup
Although there was no difference between young and aged rats with the net spikes induced by chemical stimulation, activities of nociceptors in response to chemicals (bradykinin, histamine, serotonin, and prostaglandin E2) have changed with ageing shown by a longer latency in the aged rats. Our finding is supported by previous report that latency of mechanoresponsive C fibers to 10uM bradykinin was significantly longer in the aged SD rats (Taguchi, 2010).
Also, our results showed that after chemical soup the mechanical responses are enhanced both in young and old rats. Previous report showed that local application of SP had a sensitizing effect on joint afferents in response to movements in old animals (McDougall JJ, 2007). Here, our results first time showed that this sensitization was more prominent in young rats than old rats, which was evidenced by stronger enhanced mechanical responses in young rats. We found that percentages of changes in firing rates induced by inflammatory soup were higher in young rats than in aged rats. Also the increased firing could be seen in all mechanical stimulation phases including s1 to s6, where in aged rats, it was only seen in s4.
One reason for a longer latency of inflammatory mediator induced response and weakened sensitization in senescent skin might result from the reduced expressions of receptor molecules and transducers such as TRPV1, bradykinin receptors, histamine receptors and serotonin receptors, prostaglandin receptors. Indeed, in rat spinal cord, study using quantitative immunohistochemistry for serotonin (5-‐HT) and tyrosine hydroxylase (TH) in male Wistar rats of 3 and 24 months revealed significant age-‐associated declines in the monoaminergic innervation (Ranson, R. N.,2003, Age-‐associated changes in the monoaminergic innervation of rat lumbosacral spinal cord. Brain Res). In the dorsal root ganglia of aged rats, SP-‐like immunoreactivity significantly reduced compared to young adults (Bergman, 1996). Although there are no study available as for the age-‐related changes of bradykinin, serotonin and prostaglandin E2 expressions in aged rats, it has been shown that TRPV1 expression in peripheral nerve is lower in aged mice (Wang s, neurobiol Aging, 2006). This created a possibility that reduced TRPV1 expression with ageing might lead to decreased bradykinin-‐evoked and prostaglandin-‐evoked nociceptor excitation and bradykinin-‐induced mechanical hyperalgesia.
Bradykinin is produced in response to tissue injury, inflammation, or ischemia and binds to PLC coupled (BK2) receptors on sensory neurons (McMahon et al., 2006). Bradykinin elicits acute pain through immediate excitation of nociceptors, followed by a longer lasting sensitization to thermal and
mechanical stimuli (Dray and Perkins, 1993). Genetic and electrophysiological studies suggest that bradykinin-‐evoked thermal hypersensitivity is produced through PLC-‐mediated potentiation of TRPV1 (Cesare et al., 1999; Chuang et al., 2001; Premkumar and Ahern, 2000). Several studies have suggested that TRPV1 is essential to the BK-‐evoked responses (Shin et al., 2002; Ferreira et al., 2004, Neurosci Res. 2008 Katanosaka K). In addition, histamine-‐dependent itch is mediated by a subset of C-‐fiber afferents that express TRPV1 and the histamine receptor (Shim WS, 2007. TRPV1 mediates histamine-‐induced itching via the activation of phospholipase A2 and 12-‐lipoxygenase. J. Neurosci.).
Prostaglandins (PGs), another class of fatty acid derivatives, are produced at sites of inflammation and mediate inflammatory responses and sensitization by a variety of mechanisms. Protein kinase C (PKC) and PKA downstream of prostaglandin E2 receptors, sensitize/activate multiple molecules including transient receptor potential vanilloid-‐1 (TRPV1) channels, purinergic P2X3 receptors, and voltage-‐gated calcium or sodium channels in nociceptors, leading to hyperalgesia (Biol Pharm Bull. 2011,Prostaglandin E2 and pain-‐-‐an update. Kawabata A).
Recently it was shown that inflammatory mediators such as prostaglandin-‐E2 or bradykinin cause hyperalgesia by activating cellular kinases that phosphorylate TRPV1, a process that relies on a scaffolding protein, AKAP79, to target the kinases to TRPV1(J Neurosci. Btesh J, 2013). We speculated that reduced TRPV1 expression with ageing could lead to reduced bradykinin-‐evoked and prostaglandin-‐evoked nociceptor excitation and bradykinin-‐induced mechanical hyperalgesia. Also the histamine induced C-‐fiber excitation might decrease with aging since TRPV1 expressions are decreased with aging. One can speculate that the ageing effects on the structure of other ion channels such as TRPA1, could contribute to the age-‐related chemical responses. Interestingly, a study showed that the mechanosensitivity of mouse colon afferent fibers and their sensitization by inflammatory mediators require TRPV1 and ASIC 3 (J Neurosci. 2005 Jones RC 3rd). And combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization by inflammatory soup was also significantly attenuated (Kiyatkin ME, 2013).However, whether this also applied to aged cutaneous afferents needs to be investigated in the future.
References
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