1Department of Preventive Science, 2Department of Oral Science, and 3Department of Psychiatry, Schools of Dentistry and Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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Khasabov, Sergey G.,
David M. Cain,
Dinh Thong,
Patrick W. Mantyh, and
Donald A. Simone.
Enhanced Responses of Spinal Dorsal Horn Neurons to Heat and Cold
Stimuli Following Mild Freeze Injury to the Skin.
J. Neurophysiol. 86: 986-996, 2001.
The effects
of a mild freeze injury to the skin on responses of nociceptive dorsal
horn neurons to cold and heat stimuli were examined in anesthetized
rats. Electrophysiological recordings were obtained from 72 nociceptive
spinal neurons located in the superficial and deep dorsal horn. All
neurons had receptive fields (RFs) on the glabrous skin of the hindpaw,
and neurons were functionally divided into wide dynamic range (WDR) and
high-threshold (HT) neurons. Forty-four neurons (61%) were classified
as WDR and responded to both innocuous and noxious mechanical stimuli
(mean mechanical threshold of 12.8 ± 1.6 mN). Twenty-eight
neurons (39%) were classified as HT and were excited only by noxious
mechanical stimuli (mean mechanical threshold of 154.2 ± 18.3 mN). Neurons were characterized for their sensitivity heat (35 to
51°C) and cold (28 to 12°C) stimuli applied to their RF. Among
WDR neurons, 86% were excited by both noxious heat and cold stimuli,
while 14% responded only to heat. For HT neurons, 61% responded to
heat and cold stimuli, 32% responded only to noxious heat, and 7%
responded only to noxious cold. Effects of a mild freeze injury
(
15°C applied to the RF for 20 s) on responses to heat and
cold stimuli were examined in 30 WDR and 22 HT neurons. Skin freezing
was verified as an abrupt increase in skin temperature at the site of
injury due to the exothermic reaction associated with crystallization.
Freezing produced a decrease in response thresholds to heat and cold
stimuli in most WDR and HT neurons. WDR and HT neurons exhibited a mean decrease in response threshold for cold of 9.0 ± 1.3°C and
10.0 ± 1.6°C, respectively. Mean response thresholds for heat
decreased 4.0 ± 0.4°C and 4.3 ± 1.3°C in WDR and HT
neurons, respectively. In addition, responses to suprathreshold cold
and heat stimuli increased. WDR and HT neurons exhibited an 89% and a
192% increase in response across all cold stimuli, and a 93 and 92%
increase in responses evoked across all heat stimuli, respectively. Our results demonstrate that many spinal neurons encode intensity of
noxious cold as well as noxious heat over a broad range of stimulus
temperatures. Enhanced responses of WDR and HT neurons to cold and heat
stimuli after a mild freeze injury is likely to contribute to thermal
hyperalgesia following a similar freeze injury in humans.
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INTRODUCTION |
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Unlike most somatosensory
modalities, relatively little is known regarding the neural mechanisms
that underlie the sensations of cold pain and cold-evoked hyperalgesia
following injury. Previous studies of the spinal cord have demonstrated
that many nociceptive neurons, both wide dynamic range (WDR) and
high-threshold (HT) cells in the superficial and deep dorsal horn, are
excited by cold stimuli and can, in addition to encoding the intensity
of noxious heat stimuli, respond to noxious cold stimuli to 0°C
(Craig and Bushnell 1994; Craig and Serrano
1994
; Dado et al. 1994
; Ferrington et al.
1987
; Kenshalo et al. 1982
). However, the
capacity of WDR and HT neurons to encode the intensity of noxious cold
over a wide range of stimulus temperatures, including those below
0°C, has not been explored. Two lines of evidence support the
hypothesis that dorsal horn neurons can encode the intensity of cold at
temperatures well below 0°. First, intensity-dependent c-Fos
expression is observed in the superficial and deep dorsal horn of the
rat spinal cord in response to cold stimuli of approximately
15°C
and below applied to the hindpaw (Abbadie et al. 1994a
;
Doyle and Hunt 1999
). Second, subpopulations of A
and
C-fiber cutaneous nociceptors in the rat are excited by noxious cold
(LaMotte and Thalhammer 1982
; Saumet et al.
1985
) and can encode the intensity of cold at stimulus
temperatures well below 0°C (Simone and Kajander 1996
, 1997
). We therefore hypothesized that this encoding
capacity is conserved in response properties of nociceptive spinal
neurons to noxious cold stimuli extending well below 0°C.
Hyperalgesia, defined as a decrease in pain threshold and increased
pain to normally painful stimuli, often follows tissue injury and
inflammation. Although hyperalgesia is mediated in part by
sensitization of nociceptors at the site of injury, sensitization of
spinal dorsal horn neurons also contributes to hyperalgesia (see
reviews; Baranauskas and Nistri 1998; Millan
1999
; Treede et al. 1992
). Hyperalgesia to cold
as well as to heat and mechanical stimuli occurs following a mild
freeze injury to the skin (Beise et al. 1998
;
Kilo et al. 1994
; Lewis and Love 1926
).
Interestingly, freeze injury to the skin evokes a well-defined
sensation of pricking/stinging pain at the time of freezing, and the
ensuing cold hyperalgesia is characterized by decreased cold pain
threshold and a change in the quality of pain from a cold aching
sensation to a burning sensation. It is unknown whether responses of HT
and WDR neurons evoked by cold stimuli are enhanced following freeze
injury. Therefore the second aim of this study was to determine the
response characteristics of nociceptive spinal neurons at the time of
cold injury to the skin and whether responses evoked by cold and heat
stimuli are facilitated following a freeze injury. Preliminary results
have been reported (Khasabov et al. 1999
).
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METHODS |
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Subjects
Fifty-seven adult male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 290-470 g were housed in pairs on a 12:12 h light-dark cycle. Food and water were provided ad libitum. All animal procedures were approved by the Animal Care Committee at the University of Minnesota, and experiments were conducted according to the guidelines set forth by the International Association for the Study of Pain.
Surgical preparation
Rats were anesthetized by injection of ketamine (100 mg/kg) and
acepromazine (45 mg/kg). The trachea was cannulated to provide unobstructed ventilation, and a catheter was inserted into the external
jugular vein for supplemental anesthesia with pentobarbital sodium
(5-10 mg · kg1 · h
1). Areflexia was maintained by monitoring the
corneal reflex at frequent intervals throughout the experiment. Also,
there were no withdrawal responses to mild pinching of the hind paw.
The carotid artery was cannulated and blood pressure was monitored with
a pressure transducer. Experiments were terminated if mean pressure
dropped below 60 mmHg. Core body temperature was maintained at
37-38°C by a feedback-controlled heating pad. After shaving the hair
over the midline of the animal's back, an incision was made preceding
a blunt dissection of the muscles and connective tissue to expose the
vertebral column. The animal was secured within a stereotaxic frame,
and the lumbar enlargement was exposed by laminectomy. The spinal cord
was continually bathed in a pool of warm (37°C) mineral oil.
Electrophysiological recording
Extracellular recordings of single dorsal horn neurons with receptive fields (RFs) located on the plantar surface of the hindpaw were obtained using stainless steel microelectrodes (Frederick Haer, Brunswick, ME). Recording electrodes were lowered into the spinal cord using an electronic micromanipulator (Burleigh) in 5-µm steps. Recordings were made only from single neurons whose amplitude could be easily discriminated. Electrophysiological activity was amplified, audio-monitored, and displayed on an oscilloscope before being sent to a computer for data collection using a customized version of Lab View (National Instruments, Austin, TX) software that enabled storage of raw data, discriminated impulses, and stimulus temperature.
Functional classification of spinal neurons
Search stimuli consisted of mechanical stimulation (stroking the skin and mild pinching with the experimenter's fingers) of the rat hindpaw. The RFs of isolated neurons were mapped with a supratheshold von Frey monofilament. Each spinal neuron was characterized based on its response to graded intensities of mechanical stimulation applied to the RF. Innocuous stimuli consisted of stroking the skin with a cotton swab. Noxious stimulation included mild pinching with the experimenter's fingers and with serrated forceps, but this latter stimulus was applied sparingly to avoid neuronal sensitization. Neurons were classed functionally according to responses evoked by mechanical stimuli as 1) low-threshold (LT) if they were excited maximally by innocuous stimulation, 2) wide dynamic range (WDR) if they responded in a graded fashion to increasing intensity of stimulation, and 3) high-threshold (HT) if responses were evoked by noxious stimulation only. Only WDR and HT neurons were studied.
Thermal stimulation
All thermal stimuli were applied by a feedback-controlled Peltier device (contact area of 1 cm2) to RFs located on the plantar surface of the hindpaw. A series of nine heat stimuli from 35 to 51°C (each with a duration of 5 s) was delivered in ascending steps of 2° increments. The rise/fall rate for each heat stimulus was 18°C/s. An interval of 60 s occurred between each pair of heat stimuli.
A series of 11 cold stimuli was applied in descending increments of
4° from 28 to 12°C. Cold stimuli were each delivered with a
rise/fall rate of 5°C/s and applied for 10 s. The interstimulus interval was 180 s. The baseline temperature for both heat and cold stimuli was 32°C.
Experimental design
Following identification and general functional characterization
of a neuron as WDR or HT, the RF was mapped by stroking and mildly
pinching with forceps and outlined on the skin with a felt-tip pen.
Mechanical threshold (mN) was determined using calibrated von Frey
monofilaments applied to the most sensitive area of the RF. Before the
freeze injury, heat stimuli were applied first followed after 10 min by
cold stimuli. At 10 min following freeze injury produced by 15°C
applied for 20 s, cold stimuli and then heat stimuli were
reapplied to the RF. Freezing of the skin was documented by an abrupt
increase in skin temperature at the site of injury due to the
exothermic reaction associated with crystallization (Beise et
al. 1998
). In most experiments one neuron was studied on each
side of the spinal cord. After each cell was studied the recording site
was marked by passing current (20 µA for 20 s) through the
recording electrode.
Histology
At the end of each experiment, animals received an overdose of pentobarbital sodium and were perfused with normal saline followed by 10% Formalin containing 1% potassium ferrocyanide. Serial transverse sections (50 µm) were stained with neutral red. Recording sites were identified by Prussian Blue marks or by small lesions.
Data analysis
Electrophysiological responses were recorded on videotape and analyzed off-line with the use of a customized LabView software and a computer interface. Discriminated responses and raw spike data were stored as retrievable events on a laboratory computer. To assess responses evoked by thermal stimuli, spontaneous activity was measured for a period of 10 s before each thermal stimulus and was subtracted from the response evoked during (and for 10 s after) each thermal stimulus. Comparisons between WDR and HT neurons in mean response thresholds for mechanical, cold, and heat stimuli were made using Student's t-tests. Paired t-tests were used to determine the effect of cold injury on response thresholds of WDR and HT neurons. Responses evoked by suprathreshold cold and heat stimuli before and after injury were compared using two-way ANOVAs with repeated measures. Post hoc comparisons between mean responses evoked by individual stimulus temperatures before and after injury were made using the Tukey test. For each neuron, the total sum of impulses evoked by the full range of heat or cold stimuli was calculated. The mean sums of impulses of WDR and HT neurons evoked by heat or cold stimulation were compared using paired t-tests.
To further determine effects of cold injury on stimulus-response relationships for heat and cold stimuli, power functions were generated for each neuron before and after injury. Thermal stimuli were normalized by defining a "zero point" as the highest stimulus temperature that did not evoke a response. Stimulus intensities were defined as the difference in intensity from the zero point. For example, since cold stimuli were delivered in increments of 4°C, the threshold intensity was assigned an intensity of 4°C, and the subsequent stimulus was assigned an intensity of 8°C, and so on. After log-log transformation, slopes of stimulus-response functions between WDR and HT neurons before and after injury were compared using Student's t-tests. A probability value of 0.05 was considered significant for all statistical analyses. Data are presented as means ± SE unless stated otherwise.
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RESULTS |
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General characteristics of WDR and HT neurons
A total of 72 thermal-sensitive dorsal horn neurons with RFs located on the glabrous surface of the hindpaw were selected for study. Forty-four neurons (61%) were classified as WDR and were excited by both innocuous and noxious mechanical stimuli, while 28 neurons (39%) were classified as HT and excited only by noxious mechanical (pinch) stimuli. The mean mechanical threshold of 12.8 ± 1.6 mN (mean ± SE; range, 0.4-30.1 mN) for WDR neurons was significantly lower than the mean threshold of 154.2 ± 18.3 mN (44.1-431.2 mN) for HT neurons (P < 0.001).
Spontaneous activity was exhibited by 25 of 44 WDR neurons (56.8%) at a rate ranging from 0.1 to 3.2 Hz. Only 3 of 28 HT neurons (10.7%) were spontaneously active (0.1-1.1 Hz).
All 72 neurons were excited by heat and/or cold stimuli. However, there were differences between cell types in their proportion excited by heat and cold (Table 1). With the use of a wide range of stimulus temperatures, it was found that 86.4% (38 of 44) of WDR cells were excited by both noxious heat and noxious cold stimuli, whereas only six WDR neurons (13.6%) were only sensitive to heat. No WDR neurons were excited by cold stimuli but not to heat. As for HT neurons, 60.7% (17 of 28) responded to both heat and cold stimuli, and 9 of 28 HT neurons (32.1%) were excited only by noxious heat. The remaining two HT neurons (7.2%) were excited only by noxious cold and not by noxious heat.
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Recording sites of 21 neurons (12 WDR and 9 HT) were identified histologically. Each of these neurons was excited by both heat and cold stimuli, and their recording sites were distributed throughout the dorsal horn in both superficial and deep laminae. The schematic in Fig. 1 illustrates the location of recording sites for WDR and HT neurons.
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Response characteristics of WDR and HT neurons to thermal stimuli
RESPONSE THRESHOLDS.
Mean responses to cold and heat stimuli were obtained only from those
neurons that were sensitive to thermal stimulation. As illustrated in
Fig. 2, the mean response threshold of
WDR neurons for cold differed significantly from that of HT neurons
(Fig. 2A). The mean cold threshold for 38 WDR neurons was
14.6 ± 1.3°C (range: 28 to 0°C) compared with 0.6 ± 1.1°C (range: 8 to
12°C; P < 0.001) for 19 HT
neurons. The greater sensitivity of WDR neurons to cold is further
illustrated in Fig. 2B, which shows the cumulative proportion of WDR and HT neurons excited by each cold stimulus. Although 71% of WDR neurons were excited by 12°C, none of the HT
neurons responded to this temperature. Nearly all (97%) WDR neurons
were excited by stimulus temperatures >0°C, and 100% of these
neurons were excited by a stimulus temperature of 0°C. In contrast,
only 26% of HT neurons were excited by stimulus temperatures >0°C.
Furthermore, 0°C excited only 58% of HT neurons, and 100% of these
neurons responded only when the stimulus temperature reached
12°C.
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RESPONSES TO SUPRATHRESHOLD STIMULI.
Figure 3 shows a representative example
of the responses of a WDR and a HT neuron to cold and heat stimuli. In
general, neurons encoded the intensity of cold and heat stimuli;
however, WDR neurons exhibited lower response thresholds to cold
stimuli than did HT neurons. As can be seen from Fig.
4A, cold evoked a greater
number of impulses from WDR (n = 38) than from HT
(n = 19) neurons. A two-way ANOVA with repeated
measures revealed significant differences in stimulus-response curves,
which plot the number of impulses evoked by each cold stimulus, of WDR
and HT neurons to stimulus temperatures between 28 and 12°C
(P < 0.01). For example,
4°C evoked a mean of
139.8 ± 27.7 impulses/response in WDR neurons, but only 31.3 ± 8.9 impulses/response in HT neurons. A significant difference
between WDR and HT neurons was also found in the average cumulative sum
of impulses evoked by all stimulus temperatures (Fig. 4B).
The mean number of cumulative impulses for WDR neurons was 829.6 ± 141.0 impulses, whereas HT neurons exhibited a mean cumulative total
of 320.4 ± 97.7 impulses to all cold stimuli (P < 0.004). Thus the mean cumulative response of WDR neurons evoked by
all cold stimuli was 159% greater than the cumulative response of HT
neurons.
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Responses of WDR and HT neurons during skin freezing
The effects of a mild cold injury to the skin, produced by
15°C applied for 20 s, were studied for 30 WDR and 22 HT
neurons. Skin freezing was verified by the sudden increase in skin
temperature, measured at the skin-thermode interface, that occurs
during the process of fluid crystallization. We consider the freeze
injury mild since it produced erythema but not blistering of the skin. Freezing occurred between 0.7 and 6 s after the thermode reached a
temperature of
15°C and resulted in an abrupt, high-frequency discharge of all neurons tested. A representative response of an
individual WDR neuron evoked during freezing is provided in Fig.
5. This neuron was excited by noxious
cold prior to skin freezing and exhibited a high-frequency discharge at
the onset of freezing. Following injury, spontaneous activity developed in neurons that were quiescent prior to injury, and the rate increased in those neurons that had exhibited spontaneous activity before freezing occurred. The increase in spontaneous activity persisted for
5-60 min. Mean discharge rates of WDR and HT neurons increased significantly during the first 10 s immediately following the onset of skin freezing as compared with the level of spontaneous activity prior to freezing (P < 0.03). Mean discharge
rates of WDR and HT neurons increased from 0.9 ± 0.4 to 53.3 ± 9.7 Hz and from 0.4 ± 0.3 to 31.0 ± 7.1 Hz,
respectively. Moreover, mean discharge rates of evoked by freezing were
higher for WDR neurons (P < 0.04).
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Effect of mild freeze injury on responses evoked by thermal stimuli
RESPONSE THRESHOLDS. Skin freezing increased the thermal sensitivity for each of the 25 WDR neurons and each of the 18 HT neurons. As illustrated in Fig. 6, response threshold to cold stimuli decreased after injury. For WDR neurons, mean cold threshold decreased from 12.2 ± 1.5°C to 20.6 ± 1.4°C (P < 0.001). For HT neurons, cold threshold decreased from 0.3 ± 1.5°C, to 10.6 ± 1.9°C (P < 0.001). The average decrease in cold thresholds was 9.0 ± 1.3°C for WDR neurons and 10.0 ± 1.6°C for HT neurons. For individual WDR and HT neurons the range of threshold change was 4-20°C.
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RESPONSES TO SUPRATHRESHOLD STIMULI.
Figure 8 compares the responses of a
single WDR neuron to cold (20, 8, and 12°C) and heat (39, 43, and
51°C) stimuli before and after injury. After freeze injury the
typical changes observed were a decrease in thermal thresholds and an
increase in the number of impulses to suprathreshold stimuli. As shown
in Fig. 9A, the average number
of impulses of WDR neurons to suprathreshold cold stimuli increased
after cold injury (P < 0.02). Figure 9B
shows that WDR neurons exhibited an 89% increase in the mean
cumulative sum of evoked action potentials across all cold stimuli
after injury (P < 0.001).
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Newly acquired responses to thermal stimuli following freeze injury
All neurons described above that developed enhanced responses to
cold and heat stimuli after freeze injury were excited by both cold and
heat stimuli prior to injury. However, we also studied the effects of
freeze injury on five neurons (3 WDR and 2 HT) that were initially
sensitive to heat but not to cold stimuli (down to 12°C). Each of
these neurons was excited by cold only at the time of skin freezing,
but not before, and freeze-evoked discharge rates were similar to those
of cold-sensitive neurons. A representative example is provided in Fig.
11A. The response occurred
at the onset of freezing, which is indicated by the sudden rise in skin
temperature. After injury, each neuron was excited by cold stimuli,
with response thresholds ranging from 24 to
8°C (mean threshold:
11.2 ± 4.6°C). Responses of each neuron evoked by cold stimuli
after injury are provided in Fig. 11B. Responses to heat
also increased and were similar to the increased responses to heat
described above. Mean response threshold decreased from 45.0 ± 1.3°C before injury to 41.4 ± 1.6°C after injury
(P < 0.02), and responses evoked by suprathreshold
heat stimuli increased (data not shown).
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In addition to the 72 neurons that were excited by thermal stimulation of the skin, we also determined whether freeze injury could elicit novel responses from nociceptive neurons that were not excited by heat and cold stimuli prior to injury. Four such cells (2 WDR and 2 HT) were tested, none of which developed sensitivity to cold or to heat. Although these neurons were excited during skin freezing, their mean discharge rate was much lower (14.7 ± 7.5 Hz) than those of thermosensitive neurons. In addition, responses evoked by freezing were transient and lasted approximately 1 min.
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DISCUSSION |
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Proportion and location of spinal neurons excited by noxious thermal stimuli
Our results indicate that a majority of the nociceptive spinal
dorsal horn neurons that encode mechanical stimulus intensity in rats
are excited by and encode the intensity of noxious heat. This finding
agrees with earlier studies of the rat dorsal horn (Menetrey et
al. 1979; Mitchell and Hellon 1977
) and with
similar results from spinothalamic tract (STT) neurons (Dado et
al. 1994
; Douglass and Carstens 1997
) and from
lamina I spinoparabrachial (SPB) neurons (Bester et al.
2000
). The data are also in agreement with results obtained
from STT neurons in cats (Christensen and Perl 1970
;
Craig and Kniffki 1985
; Henry 1976
) and
monkeys (Ferrington et al. 1987
; Kenshalo et al.
1979
; Price et al. 1978
).
Earlier studies of the responses of nociceptive dorsal horn neurons to
innocuous or noxious cold stimuli (Christensen and Perl
1970; Craig and Kniffki 1985
; Menetrey et
al. 1979
) utilized methods in which stimulus intensity either
could not be precisely controlled (e.g., evaporation of ethyl chloride,
application of ice), or in which a more restricted range of stimulus
intensities was delivered. Studies using thermodes with Peltier devices
for precise control of stimulus temperature enabled quantitative
analysis of response thresholds to noxious cold to 0°C and
demonstrated that many nociceptive dorsal horn neurons encoded
intensity of noxious cold, at least to 0°C (Craig and Bushnell
1994
; Craig and Serrano 1994
; Dado et al.
1994
; Ferrington et al. 1987
; Kenshalo et
al. 1982
). We have extended previous results by delivering controlled stimulus intensites in discrete stimulus increments to as
cold as
12°C. Using this paradigm, we found that cold thresholds of
HT neurons can extend to
12°C (compared with 0°C for WDR
neurons), and that the majority of WDR (86.4%) and HT (60.7%) neurons
were responsive to noxious heat as well as to noxious cold stimuli. Another two HT neurons (7.1%) were sensitive to noxious mechanical and
cold stimuli, but not to noxious heat. In addition, it was recently
reported that approximately 35% of SPB neurons responsive to heat were
also excited by and encoded the intensity of noxious cold at stimulus
temperatures below 0°C (Bester et al. 2000
). Thus
using cold stimuli with intensities below 0°C is necessary to fully
characterize the proportion of neurons excited by cold and reflects
findings of response thresholds for cold and encoding of stimulus
intensities below 0°C among many cutaneous A
and C-fiber
nociceptors (Simone and Kajander 1996
,
1997
).
The use of cold stimuli at intensities below 0°C is also required to
evoke Fos-like immunoreactivity in the spinal cord by cold
(Abbadie et al. 1994a; Doyle and Hunt
1999
). Stimuli of approximately
15°C and below produced
labeling for c-Fos in the superficial (laminae I-II) and deep (laminae
III-VI) dorsal horn and were intensity dependent. Interestingly,
noxious heat stimulation of the hind paw induced expression of c-Fos in
the spinal cord with a distribution similar to that produced by noxious
cold stimuli (Abbadie et al. 1994b
), suggesting that
many dorsal horn neurons may process information from heat- and
cold-sensitive nociceptors. Collectively, these findings suggest that
dorsal horn neurons encode the intensity of cold at temperatures well
below 0°C and that earlier studies may have underestimated the
proportion of spinal dorsal horn neurons that are excited by noxious cold.
Similar to previous reports, the recording sites of WDR and HT neurons
were located throughout the dorsal horn including in both superficial
and deep dorsal horn (Burstein et al. 1991; Dado et al. 1994
). No relationship could be discerned between the
observed response properties of WDR and HT neurons and their location
in the dorsal horn, which is consistent with similar results obtained from lumbar dorsal horn neurons in rats (Menetrey et al.
1979
), lumbar STT neurons in monkeys (Kenshalo et al.
1979
), and from cervical STT cells in rats (Dado et al.
1994
). HT neurons that were responsive to noxious heat and to
noxious cold may correspond to the STT neurons found in lamina I of
cats that are responsive to noxious heat, pinch, and cold stimuli (HPC
cells) (Han et al. 1998
). In the present study, these
cells were found in the superficial as well as the deep dorsal horn,
although we do not know whether any were projection neurons.
Of 72 thermal-sensitive nociceptive neurons, 21% were exclusively
heat-sensitive neurons, and only 3% were exclusively cold-sensitive neurons. This difference may reflect psychophysical results reporting a
greater ability to detect and discriminate small changes in the
intensity of noxious heat compared with noxious cold, and a steeper
slope of stimulus-intensity functions for heat relative to cold
stimulation (Morin and Bushnell 1998). However, the
number of neurons that did not respond to cold stimuli may have been underestimated since some A
nociceptors in rat skin have cold response thresholds as low as
18°C (Simone and Kajander
1997
). We did not use cold stimulus temperatures lower than
12°C because of possible skin freezing and sensitization.
Comparison of responses of dorsal horn neurons to noxious heat and cold stimuli
For both WDR and HT neurons, differences were observed in terms of the temperature-response curve, cumulative number of impulses, cumulative number of neurons responding to each temperature, and thermal thresholds. However, for both groups of neurons, the magnitudes of these differences were greater in their responses to cold than to heat.
Regarding heat sensitivity, WDR neurons exhibited a small but
significantly lower mean response threshold, and greater discharge of
impulses to suprathreshold stimuli than did HT neurons. These results
agree with previous reports in rats (Bester et al. 2000), cats
(Craig and Serrano 1994
), and monkeys (Bushnell et al.
1984
; Ferrington et al. 1987
), whereas other
studies found no significant differences between WDR and HT neurons in
their responses to heat (Dado et al. 1994
;
Kenshalo et al. 1979
; McHaffie et al.
1994
; Surmeier et al. 1986
). We found that the
mean slopes of the power functions for both WDR and HT heat-sensitive
neurons were nearly identical, suggesting that these neurons encode
stimulus intensity similarly.
Regarding cold stimulation, we observed a greater difference between
WDR and HT neurons. Nearly one-half (42.1%) of WDR but none of the HT
neurons had response thresholds for cold in the innocuous range
(20°C). Also, the mean cold threshold for WDR neurons was lower
(response to higher stimulus temperatures) than the mean threshold of
HT neurons (14.6 vs.
0.6°C). In addition, the evoked number of
impulses as a function of stimulus intensity was greater for WDR
neurons. However, the slopes of power functions that related the number
of evoked impulses to stimulus intensity were greater for HT neurons.
One interesting observation is that the difference between WDR and HT
in their sensitivity to cold stimuli is consistent with differences in
responses to cold of subpopulations of cutaneous nociceptors. We found
that C-fiber nociceptors were more sensitive to cold (lower response
threshold) than A
nociceptors (Simone and Kajander
1996
, 1997
). Most A
nociceptors thresholds
for cold occurred near or below 0°C. Response thresholds of A
nociceptors were significantly higher (colder stimulus temperatures)
than thresholds of C fibers, and many exhibited response thresholds
below 0°C. Responses of both WDR and HT nociceptors increased as
stimulus temperature decreased. These data demonstrate that A
and C
nociceptors are excited by a wide range of cold stimuli and suggest
that the proportion of cutaneous nociceptors excited by noxious cold
has been underestimated in previous studies. Thus one possibility is
that WDR neurons receive more input from C-fiber nociceptors that are
sensitive to cold stimuli and perhaps also from innocuous cold-specific
fibers than HT neurons, whereas HT neurons receive more input from A
nociceptors that have high response thresholds for cold stimuli. This
is supported by psychophysical studies in humans that have shown
different qualities of cold pain sensation. The quality of cold pain
evoked by moderately noxious cold temperatures has been described as a
dull, aching pain but changes to a sharp and pricking pain as stimulus
intensity is increased (Davis 1998
; Lewis and
Love 1926
; Wolf and Hardy 1941
). Thus the change
in the quality of sensation from aching to pricking with increasing
stimulus intensity may involve the recruitment of HT neurons by colder
stimuli. The separate qualities of dull aching and pricking pain evoked
by cold stimuli might be theoretically similar to the classic first and
second pain sensations evoked by noxious heat. First pain, which is
characterized as pricking pain, has been shown to be mediated
by activation of A
nociceptors (Campbell and LaMotte
1983
), whereas second pain is characterized as a dull, burning
sensation and has been shown to be mediated primarily by activation of
C-fibers (Beitel and Dubner 1976
; Gybels et al.
1979
; Torebjörk and Hallin 1974
).
Responses of dorsal horn neurons during skin freezing
During the process of skin freezing, the interstitial fluid
crystallizes, which results in an exothermic reaction due to
condensation. The exothermic reaction can be measured objectively as a
rapid increase in skin temperature (Beise et al. 1998;
Keatinge and Cannon 1960
). Since our Peltier device
measured the temperature of the interface between the skin and the
thermode, we were able to record the increase in skin temperature
during freezing, thus providing an accurate measure of the time of skin
freezing. It was found that all WDR and HT neurons exhibited a
high-frequency discharge at the onset of skin freezing. This discharge
was not due to skin temperature per se because it never occurred in the absence of freezing, regardless of stimulus temperature. Psychophysical studies in humans have shown that the onset of skin freezing is associated with an abrupt sensation of pricking or stinging pain (Beise et al. 1998
; Granberg 1991
;
Kilo et al. 1994
; Lewis and Love 1926
).
The abrupt pricking/stinging sensation did not occur in the absence of
freezing. Our results suggest that both WDR and HT neurons contribute
to pain sensation associated with skin freezing, although WDR neurons
exhibited a greater discharge during freezing. It is not presently
known which specific subtypes of peripheral afferent fibers are excited
at the onset of freeze injury to provide the afferent drive for WDR and
HT neurons.
Sensitization of dorsal horn neurons following a mild freeze injury: potential mechanisms and relation to psychophysical studies
Our results show that a mild freeze injury to the skin enhances
activity of both WDR and HT neurons to heat and cold stimuli applied at
the site of injury. This finding of cross sensitization between
different modalities of thermal stimulation may suggest that a common
mechanism accounts for the increase in responses to heat and cold.
However, enhanced responses of WDR and HT neurons to both noxious heat
and cold were not found following repeated noxious heating of the skin
(Kenshalo et al. 1982). Rather, only heat-evoked
responses were increased (Kenshalo et al. 1979
). Our finding of cross-sensitization for heat and cold stimuli may result from properties of sensitization that are unique to cold injury to the
skin, and must be different from sensitization induced by heating.
Previous psychophysical studies of cutaneous hyperalgesia following
freeze injury have reported hyperalgesia to mechanical, heat, and cold
stimuli applied to the injured skin, although the magnitude and
duration of hyperalgesia is related to the severity of injury. A brief,
mild freeze injury similar to that used in the present study produced a
lowering of cold and heat pain thresholds soon after injury
(Beise et al. 1998; Kilo et al. 1994
).
More severe freeze injuries (e.g., produced by
28°C) also produced mechanical hyperalgesia both within and surrounding the injured skin
(secondary hyperalgesia) that developed several hours after injury and
persisted for 24 h or more (Kilo et al. 1994
;
Lewis and Love 1926
). In the present study we did not
investigate whether responses evoked by mechanical stimulation
increased, nor whether the increase in responses to heat and cold
stimuli occurred in areas of the RF other than the contact area of the
Peltier device. However, our data support the view that sensitization
of both WDR and HT neurons may contribute to cold and heat hyperalgesia produced by freeze injury.
The mechanisms by which responses of dorsal horn neurons to heat and
cold stimuli are increased after freeze injury are unclear. We believe
that the enhanced responses are the result of injury and are not solely
due to cold temperature because sensitization was not observed when the
skin was cooled to 15°C but freezing did not occur. However, we do
not know whether peripheral or central mechanisms, or both, contributed
to the increased responses of dorsal horn neurons to heat and cold
stimuli after the freeze injury. One possibility is that freeze causes
sensitization of cutaneous nociceptors to heat and cold stimuli. It is
known that sensitization of nociceptors contribute to heat hyperalgesia
following heat injury to the skin (Campbell and LaMotte
1983
; LaMotte et al. 1983
; Meyer and
Campbell 1981
; Torebjörk et al. 1984
).
Whether nociceptors can become sensitized to cold stimuli following
freeze injury and thereby contribute to cold hyperalgesia needs to be determined.
A second possibility is that the increased responses to heat and cold
stimuli after freeze injury are due to central sensitization. It is
well-known that tissue injury and inflammation increase excitability of
nociceptive dorsal horn neurons whereby RFs become enlarged and
responses evoked by mechanical and thermal innocuous and noxious inputs
are enhanced (Simone 1992; Treede et al.
1992
; Woolf 1992
). High-frequency discharge in
nociceptors, particularly C-fiber nociceptors, may lead to a state of
central sensitization in which responses of dorsal horn neurons
produced by activation of low-threshold afferent fibers, including
cooling-specific fibers, as well as by nociceptors are increased. If
central sensitization occurs following a mild freeze injury, it will be
important to determine whether similar cellular events, such as
activation of N-methyl-D-aspartate (NMDA)
receptors, contribute to hyperexcitability as has been demonstrated
with other models of injury and inflammation.
A particularly interesting finding was that five heat-sensitive neurons
that did not appear to be responsive to cold stimuli before injury
developed responses to cold after injury. This phenomenon might also be
related to nociceptor sensitization or to central sensitization. In any
event, we cannot preclude the possibility that these neurons received
input from A nociceptors with cold thresholds below
12°C, as
described earlier (Simone and Kajander 1996
,
1997
), and sensitivity to cold would have been observed before injury if colder stimulus temperatures were used.
Conclusions
We have shown that the majority of WDR and HT neurons in the spinal cord are excited by and encode the intensity of noxious heat as well as noxious cold stimuli, and that both types of neurons develop increased responses to heat and cold stimuli following a mild cold damage to the skin. The decrease in response threshold and the increase in evoked response to cold stimuli following freeze injury were greater than those observed for heat stimuli. It is concluded that sensitization of both WDR and HT neurons located in the superficial as well as the deep dorsal horn contributes to thermal hyperalgesia following mild freeze injury to the skin. Sensitization of these neurons may also underlie hyperalgesia following more severe frostbite injuries.
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ACKNOWLEDGMENTS |
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We thank Dr. Glenn J. Giesler, Jr. for reading an earlier version of the manuscript.
This work was supported by National Institutes of Health Grants NS-33908 and DA-11986.
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FOOTNOTES |
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Address for reprint requests: D. A. Simone, Dept. of Oral Science, University of Minnesota, 515 Delaware St. SE, 17-252 Moos, Minneapolis, MN 55455 (E-mail: simon003{at}umn.edu).
Received 23 February 2001; accepted in final form 20 April 2001.
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REFERENCES |
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