Abstract To clarify the mechanism by which changes inchronic pain are induced by cold environments, rats ren-dered neuropathic by a chronic constriction injury (CCI)to the sciatic nerve were exposed to low ambient temper-ature (LT; 7°C decrease from 22°C) in a climate-con-trolled room. LT exposure aggravated pain-related be-haviors in CCI rats, i.e., decreased the threshold to vonFrey hair and paw pressure stimulation, prolonged theduration of foot withdrawal to pinprick stimulation, andincreased the cumulative duration of guarding posture.Lumbar sympathectomy (SYX) did not inhibit LT-in-duced augmentations of pain-related behaviors in CCIrats. LT exposure decreased the skin temperatures ofboth hind paws to the same degree in the sham-operatedcontrol and SYX rats, while in the CCI and SYX+CCIrats it caused a larger temperature decrease in the injuredpaw than in the uninjured one. These results indicate thatLT exposure augments abnormalities in pain-related be-haviors of neuropathic rats, and also suggest that sympa-thetic nervous activity is not a predominant factor in theaugmenting mechanism.

Key words Chronic constriction injury · Mechanicalallodynia · Mechanical hyperalgesia · Spontaneous pain ·Cold exposure

Introduction

Various meteorological factors, such as barometric pres-sure, temperature, humidity, rain, and storms, have beensuspected of contributing to changes in pain (Mitchell1877; Hollander 1961, 1962; Anderson et al. 1965;Sulman et al. 1970; Yunus et al. 1981; Rasker et al.

1986; Nurmikko and Bowsher 1990; Shutty et al. 1992;Hooshmand 1993; Hendler et al. 1995; Jamison et al.1995). In contrast, a recent study has thrown strongdoubt on the relationship between weather change andpain, and even suggested that psychological factors maycontribute to the belief that chronic pain is related toweather change (Redelmeier and Tversky 1996). To seekan answer to this contradiction, we have examined theeffects of lowering barometric pressure within the typi-cal range of natural weather change (20 mmHg lowerthan atmospheric pressure) on the pain-related behaviorsin rats rendered neuropathic by a chronic constriction in-jury (CCI) on the sciatic nerve (Sato et al. 1999). Ourstudies indicated that exposure to low barometric pres-sure augmented the mechanical allodynia and hyperalge-sia shown in the hind paw of CCI rats, and that sympa-thetic nervous activities play a significant role in theaugmenting mechanism. These findings thus support re-ports that approaching low-pressure systems aggravatechronic pain (Hollander 1962; Guedi and Weinberger1990; Jamison et al. 1995).

A positive relationship between low ambient tempera-ture and aggravated pain in humans has also been report-ed (Mitchell 1877; Hollander 1962; Yunus et al. 1981;Guedi and Weinberger 1990; Shutty et al. 1992;Hooshmand 1993). To date, however, there have been nocontrolled animal studies examining the mechanism thatcauses such low temperature (LT)-related pain.

In the present study, we investigated whether lower-ing the ambient temperature, simulated in a climate-controlled room, aggravates pain-related behaviors inCCI rats. Since in another recent study we found thatsympathetic activities underlie the mechanism by whichlow-pressure exposure augments the pain-related behav-iors in CCI rats, in the present study we again performedsympathectomy to see whether this was true with LT ex-posure as well. A preliminary account of this work hasbeen published in abstract form (Sato et al. 1996).

J. Sato (✉ ) · H. Morimae · K. Takanari · Y. Seino · T. OkadaM. Suzuki · K. MizumuraDepartment of Neural Regulation,Research Institute of Environmental Medicine, Nagoya University,Furo-cho, Chikusa-ku, Nagoya 464–8601, Japane-mail: jun@riem.nagoya-u.ac.jpTel.: +81-52-7893864, Fax: +81-52-7893889

Exp Brain Res (2000) 133:442–449DOI 10.1007/s002210000451

R E S E A R C H A RT I C L E

Jun Sato · Hirohumi Morimae · Keisuke TakanariYusuke Seino · Taro Okada · Minemori SuzukiKazue Mizumura

Effects of lowering ambient temperature on pain-related behaviorsin a rat model of neuropathic pain

Received: 5 July 1999 / Accepted: 12 April 2000 / Published online: 7 June 2000© Springer-Verlag 2000

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Materials and methods

The following investigations were carried out according to a pro-tocol approved by the Animal Care Committee, Research Instituteof Environmental Medicine, Nagoya University.

Experimental animals and surgeries

The experiments were performed on 107 male Sprague-Dawleyrats (200–250 g). The animals were kept in an animal room at22°C with a 12 h alternating light–dark cycle, and were given foodand water ad libitum. Rats were randomly assigned to one of fourdifferent surgical groups: (1) control rats, in which the right sciaticnerve was exposed but not ligated (n=32), (2) CCI rats, in whichthe right sciatic nerve was exposed and ligated (n=47), (3) lumbarsympathectomy (SYX) rats, in which a transperitoneal sympathec-tomy was performed (n=14), and (4) SYX+CCI rats, in which atransperitoneal sympathectomy was followed by CCI surgery(n=14).

Unilateral peripheral neuropathy was induced according to themethod described by previous authors (Bennett and Xie 1988).Briefly, under sodium pentobarbital anesthesia (60 mg/kg, i.p.),the right sciatic nerve of the CCI rats and SYX+CCI rats was li-gated at the upper thigh level with four loose ligatures using chro-mic gut (4/0). In the control rats, the right sciatic nerve was ex-posed, but no ligatures were applied.

In SYX rats and SYX+CCI rats, sympathectomy was per-formed 12–19 days before the CCI or sham surgery. After segmen-tal identification of the ganglia (Baron et al. 1988; Koltzenburg etal. 1992), both sides of the sympathetic chain from L2 to L5 werecompletely cut out. The interval between the sympathectomy andthe CCI or sham procedures was considered sufficient for thecomplete degeneration of postganglionic fibers (Andres et al.1985).

LT exposure

Before starting LT exposure, the animals were kept in the experi-mental climate-controlled room (baseline ambient temperature22°C, relative humidity 60%) for 60–90 min. The ambient temper-ature was decreased by 7°C from 22°C over 30 min and kept atthis level for 90 min, then returned to the baseline temperatureover 30 min (Fig. 1). A change of 7°C was chosen because thisvalue is within the typical range of natural weather change. Paintests and skin temperature measurements (described below) were

carried out before exposure (pre-exposure value), at the lowesttemperature (midexposure value), and after exposure (postexpo-sure value), as shown in Fig. 1. In order to measure the values onschedule, only two or three rats were exposed to LT at one time.Relative humidity was kept at 60% and barometric pressure wasnatural barometric pressure. All tests were performed on a daywhen no sudden change in the weather was expected (atmosphericpressure: 754–768 mmHg), in order to eliminate the possibility ofcontaminating influences from external atmospheric pressurechange.

Pain test procedures

Seventy rats were used for only one of the pain tests, and 12 ratswere used for both the von Frey hair (VFH) test and the pinpricktest. The investigator performing the measurements was not in-formed of the kind of surgical treatment that the rat had under-gone; however, it is impossible to blind investigators to the pres-ence of CCI surgery because of the rat's typical hind paw posture(Bennett and Xie 1988).

Mechanical allodynia

Mechanical allodynia was measured by the VFH test and the pawpressure test. For the VFH test, each rat (n=31) was placedindividually beneath an inverted transparent plastic cage(11×17×11 cm) with a wire mesh bottom. Hand-made VFHs(diameter 0.2 mm) were applied perpendicular to the plantar sur-face of the right hind paw through the wire mesh with sufficientforce to cause slight bending against the paw, and held for 2 s oruntil the rat withdrew its paw. The hairs were applied in the orderof increasing bending force (from 0.4 to 30 g), with each appliedfive times at intervals of 2–3 s to different parts of the midplantarglabrous skin. The strength of the first hair in the series thatevoked at least one positive response among the five trials wasdesignated the pain threshold.

The paw pressure (Randall-Selitto) test was performed with aUgo Basil analgesymeter (Apelex). A constantly increasing pres-sure was applied to the area between the third and fourth metatarsiof the dorsal part of the right hind paw (in the sciatic nerve territo-ry) until the rat withdrew its paw (n=13). The nociceptive thresh-old was defined as force, in grams, at which the rat withdrew itspaw. To decrease the variability in nociceptive thresholds, the ratswere trained by applying pressure at 5-min intervals for 1 h perday for 3–4 days before the CCI or sham surgery. A set of trials at5-min intervals for 1 h was also performed in the pre-, mid- andpostexposure periods. The average withdrawal threshold of thelast six trials constituted the pain threshold.

Mechanical hyperalgesia

Mechanical hyperalgesia was measured with the pinprick test. Forthis test, the rats (n=32) were placed individually on the meshfloor, as for the VFH test. The point of a safety pin was applied tothe midplantar surface of the right hind paw through the wiremesh, and the duration of the evoked hind paw withdrawal timedwith a stopwatch. The duration of the normal withdrawal reflexwas usually too short to time manually with a stopwatch, so we ar-bitrarily assigned it a duration of 0.2 s. The hind paw withdrawalsseen in the CCI rats were very much longer (usually over 2 s). Ameasurement cut-off of 15 s was applied to abnormally prolongedwithdrawals.

Spontaneous pain

For the measurement of spontaneous pain (ongoing pain withoutapparent external stimuli) in the neuropathic rats, we observed theanimals' guarding behavior (Bennett and Xie 1988) in a natural

Fig. 1 Experimental protocol for simulated low-temperature (LT)exposure. Barometric pressure was the natural atmospheric pres-sure, and relative humidity was 60%. The pinprick test, von Freyhair test, paw pressure test, and spontaneous pain and skin temper-ature measurements were performed at the times indicated by thebars

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setting without intervention from the experimenter. To this end,each CCI rat (n=18) was placed on the mesh floor, as for the VFHtest. After a 5-min adaptation, the cumulative time during the next5 min that the rat held its foot off the floor was recorded. Howev-er, foot lifts associated with locomotion or body repositioningwere not counted.

Skin temperature measurement

Thirty-nine rats were used for the skin temperature measurement;twenty-five of which were used only for this test, while the re-mainder were also employed in one of the pain tests (VFH test orpinprick test). The cutaneous hind paw temperature was measuredon the plantar surface in the region of the sciatic nerve, using anon-contact infrared thermometer (Thermo-Hunter 5149; Optex).To avoid inducing stress, hind paw temperature was measuredwithout straining the rats. Each animal was placed on the wiremesh floor as for the VFH test, and the temperature of the plantar

surface of the hind paw was measured through the wire mesh. Af-ter a 5-min adaptation, two consecutive temperature determina-tions were carried out with an interval of 5–10 s, and their valuesaveraged to represent a single measurement.

Data analysis and statistics

Results are expressed as the mean ± SEM. For the VFH test re-sults, the Friedman test was used to evaluate the influences of sur-gical procedures or exposure to the LT environment. For compari-son of each value, the Wilcoxon matched-pairs signed-ranks (Wil-coxon) test was used for a post hoc analysis. The Kruskal-Wallistest and the Mann-Whitney U-test were employed to compare thevalues of control and experimental groups.

For the paw pressure and pinprick tests, as well as for mea-surements of spontaneous pain and hind paw skin temperature, re-peated measures ANOVA was used to evaluate the influences ofsurgical procedures or exposure to the LT environment. To com-pare each value, Fisher's protected least significant difference(PLSD) test was used for a post hoc analysis.

An unpaired t-test was used to compare the values of the con-trol and experimental groups in the paw pressure test. A paired t-test was employed to compare the skin temperatures of both hindpaws. Differences were considered statistically significant at theP<0.05 level.

Results

Mechanical allodynia (VFH test)

There were no significant differences in the basal valueof VFH threshold before CCI or sham surgery amongfour groups of rats (P=0.26, Kruskal-Wallis test). The

Fig. 2A–D Augmenting effect of LT exposure on mechanical al-lodynia induced by chronic constriction injury (CCI; von Frey hairtest). The data are plotted as the cumulative number among thefour groups of rats with threshold responses to each von Frey hairin the series. Ordinate Cumulative number of rats responding, ab-scissa bending force (grams) of von Frey hairs in a logarithmicscale, pre I before CCI or sham surgery, pre II pre-LT exposure,mid during LT exposure, post post-LT exposure. Data points corre-sponding to no responders and to presympathectomy were omittedfor clarity. The midvalues in CCI and lumbar sympathectomy(SYX)+CCI rats are significantly smaller than the correspondingpre II values (P=0.03, P=0.04, respectively; Friedman test fol-lowed by a post hoc analysis). There was a significant differencein the midvalue between the control group and the CCI group(P=0.001, Mann-Whitney U-test)

control rats showed a brisk, very brief withdrawal re-sponse to the VFH stimulation prior to surgery(4.7–18 g; pre I, Fig. 2A). There was some fluctuation inthe VFH threshold, but the Friedman test showed that theresponse thresholds were not significantly changed bythe sham operation or the LT exposure (P=0.1).

After the CCI surgery [postoperative day (POD)20–22], the VFH threshold of four out of ten rats was un-changed on the day when LT exposure was done; theirthresholds were 9.2, 6.0, 4.7, and 4.7 g, respectively(Fig. 2B). In the remaining six rats, however, the thresh-old was clearly reduced after the CCI surgery, from 6.0to 4.7, 6.0 to 2.2, 4.7 to 3.0, 2.2 to 1.1, 2.2 to 1.1, and 2.2to 1.1 g, respectively. Three CCI rats showed abnormallyprolonged withdrawal responses (over 2 s), and in twoCCI rats VFH stimulation also induced licking behavior.As a whole, CCI produced a significant decrease in theVFH threshold (pre II to pre I, Fig. 2B; P=0.03, Fried-man test followed by a post hoc analysis). However, thedifference in the pre II value between the control groupand the CCI group was marginally insignificant (P=0.08,Mann-Whitney U-test). To test whether LT exposure in-duces a reduction in the VFH threshold, all of the CCIrats, irrespective of the presence of allodynia before ex-posure, were exposed to the LT environments. Interest-ingly, LT exposure reduced VFH threshold in all rats butone (the unchanged threshold of this exceptional rat wasrelatively low, at 1.1 g). A post hoc analysis (Wilcoxontest) showed that VFH thresholds during LT exposure(mid) were significantly lower than those before expo-sure (pre II, P=0.008). Moreover, the midvalue of theCCI group was significantly lower than that of the con-trol group (P=0.001, Mann-Whitney U-test). This hyper-sensitivity disappeared 90 min after returning to the nor-mal temperature (post). The present results thus indicatethat LT exposure, similar to low-pressure exposure (Satoet al. 1999), aggravates mechanical allodynia in neuro-pathic rats.

However, the results of sympathectomy were clearlydifferent from those of low-pressure exposure. Figure 2Dshows the changes in VFH threshold of SYX+CCI rats(n=6). On POD 14 (33 days after SYX; pre II), VFH(4.7–18 g) evoked withdrawal responses with thresholdsnot significantly different from the pre-CCI values (pre I)of these rats or the controls [P=0.92 (Wilcoxon test) and0.13 (Mann-Whitney U-test), respectively]. This wouldappear to indicate that sympathectomy prevented the de-velopment of mechanical allodynia after the CCI proce-dure. A similar preventive effect of sympathectomy wasalso observed in experiments on low-pressure exposure(Sato et al. 1999). In contrast to the low-pressure expo-sure, however, the VFH thresholds of SYX+CCI ratswere clearly lowered during LT exposure; that is, stimula-tion with VFH (1.1–18 g) evoked hypersensitive with-drawal responses in SYX+CCI rats (pre II vs mid;P=0.04, Wilcoxon test). This hypersensitivity disap-peared 90 min after returning to the normal temperature.

It should be noted that sympathectomy alone did notsignificantly change VFH threshold (before sympathec-

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tomy=1.6–30 g, Fig. 2C; n=6), and that LT exposure hadno effect on the response thresholds of SYX rats(P=0.58, Wilcoxon test). These results indicate that theaugmenting effect of LT exposure on mechanical allody-nia does not depend on sympathetic nerve activities.

Mechanical allodynia (paw pressure test)

To confirm the results of the VFH test, an additional pawpressure test was carried out (Fig. 3). In the control rats(n=8), neither sham operation nor LT exposure alteredthe withdrawal threshold (P=0.13, ANOVA). In the CCIrats (n=5), the mean withdrawal threshold to paw pres-sure before LT exposure was 94.0±16.4 g on POD 26and 97.0±10.2 g on POD 33 (pre, Fig. 3). Comparedwith the preoperative value (135.0±19.2 g), the values onPOD 26 and POD 33 had significantly decreased (forboth values: P<0.05, ANOVA with Fisher's PLSD test).Compared with the control value, the prevalue onPOD 33 but not on POD 26 was significantly smaller(P=0.049 and P=0.18, respectively, unpaired t-test). In-terestingly, while CCI resulted in a decreased thresholdon POD 33 only, LT exposure produced a significant de-crease on both PODs 26 and 33 (for both PODs: P<0.05,Fisher's PLSD test), confirming the results of the VFHtest described above. The midvalue of CCI rats on bothPODs was significantly smaller than that of the controlrats (POD 26: P=0.002, POD 33: P=0.005, unpaired t-test). This effect disappeared when the withdrawalthreshold was measured 90 min after returning to thenormal temperature.

Fig. 3 Augmenting effect of LT exposure on mechanical allodyniainduced by chronic constriction injury (paw pressure test). The or-dinate shows the average threshold of the nociceptive hind pawwithdrawal response evoked by a pressure applied on the injuredhind paw with an analgesymeter (mean ± SEM). BS Before CCI orsham surgery, POD 26, POD 33 26 and 33 days after the surgery,pre pre-LT exposure, mid during LT exposure, post post-LT expo-sure. LT exposure decreased the threshold of the withdrawal re-sponse in the CCI rats (n=5), but not that in the control rats (n=8),on both postoperative days. +P<0.05, *P<0.05, compared with BSand each pre-exposure value, respectively (repeated measuresANOVA with a post hoc analysis). #P<0.05, compared to eachcontrol value (unpaired t-test)

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Mechanical hyperalgesia (pinprick test)

On POD 10 when the effects of LT exposure were evalu-ated, all CCI rats (n=10) showed prolonged withdrawalduration, and thus were hyperalgesic to the pinprickstimulation (pre: CCI, Fig. 4). LT exposure induced afurther increase in the withdrawal duration (pre vs mid;P<0.05, ANOVA with Fisher's PLSD test), indicating LTexposure intensifies not only mechanical allodynia butalso mechanical hyperalgesia. This augmenting effectdisappeared when the withdrawal duration was measured90 min after returning to 22°C (post).

In contrast to the results of a low-pressure exposurestudy (Sato et al. 1999), sympathectomy did not inhibitthe augmenting effect of LT exposure on the response topinprick stimulation. SYX+CCI rats (n=10) were hyper-algesic on POD 11 (23 days after sympathectomy) whenthe effects of LT exposure were evaluated (pre:SYX+CCI, Fig. 4), demonstrating that sympathectomydid not prevent the development of hyperalgesia. Thiswas in contrast to observations on the VFH threshold(Fig. 2D). In these ten rats, withdrawal duration duringLT exposure (mid) was significantly prolonged com-pared with the pre-exposure value (P<0.05, ANOVAwith Fisher's PLSD test). This hypersensitivity disap-peared 90 min after returning to 22°C (post). Sympathec-tomy itself did not induce any change in pinprick with-drawal reflex before or during LT exposure (SYX,Fig. 4; n=6; 19 days after sympathectomy).

These results indicate that, similar to mechanical allo-dynia, aggravation of mechanical hyperalgesia inducedby LT exposure does not depend on sympathetic nerveactivities.

Spontaneous pain

Differing from the sham-operated control rats, all of theCCI rats (n=18) often held the hind paw on the nerve-injured side off the floor. Because a preliminary observa-tion indicated that the foot lifting duration was maximalabout 1 week after sciatic nerve constriction, the effectsof LT exposure were evaluated on POD 6–7. Figure 5shows the change in cumulative duration of foot liftingduring LT exposure. LT exposure significantly increasedthe cumulative duration of foot lifting of the injured paw(pre vs mid, P<0.05, ANOVA with Fisher's PLSD test)but not of the uninjured one. This effect disappeared af-ter returning to 22°C. These results indicated that LT ex-posure aggravates not only stimulus-evoked pain (me-chanical allodynia and hyperalgesia), but also spontane-ous pain.

Skin temperature

In the control rats (14 days after sham operation), thetemperatures of the injured (R) and uninjured (L) hindpaws of each rat were nearly always about the same be-fore LT exposure (control-pre, Table 1). Fourteen daysafter sympathectomy, the skin temperature was slightlyhigher on the injured side in the SYX group, but the dif-ference (R-L: ∆T) was not significant (SYX-pre: P=0.54,paired t-test). Abnormally large asymmetry was ob-served in some CCI rats: the temperature on the injuredside was higher by more than 0.7°C in four rats and low-er by more than 0.7°C in two rats. On average, ∆T in theCCI group was slightly positive on POD 20–22, but thisdifference was not significant (CCI-pre, P=0.16, pairedt-test). On POD 18 (30 days after sympathectomy), the

Fig. 4 Augmenting effect of LT exposure on mechanical hyperal-gesia induced by chronic constriction injury (pinprick test). Thefigure shows the average durations of the nociceptive withdrawalresponse evoked by pinprick applied to the midplanter area of theinjured hind paw (s, mean ±SEM). Pre Pre-LT exposure, mid dur-ing LT exposure, post post-LT exposure. *P<0.05, compared witheach pre-exposure value (repeated measures ANOVA followed bya post hoc analysis). LT exposure increased the withdrawal dura-tion in both the SYX+CCI (n=10) and CCI rats (n=10), but notthat in the control (n=6) and SYX rats (n=6)

Fig. 5 LT exposure increased the spontaneous pain in the injuredpaw of CCI rats. The figure shows the change in cumulative dura-tion of foot lifting during a 5-min measurement (n=18, mean ±SEM). Pre Pre-LT exposure, mid during LT exposure, post post-LT exposure. LT exposure significantly increased the cumulativeduration of foot lifting of the injured paw, but not of the uninjuredpaw. *P<0.05, compared with the pre-exposure value (repeatedmeasures ANOVA followed by a post hoc analysis)

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skin temperature on the injured side was also higher inthe SYX+CCI group (positive ∆T), but again this differ-ence was not significant (SYX+CCI-pre, P=0.13, pairedt-test). Thus, no difference in the leg temperature on ei-ther side was found among any of the groups of rats be-fore LT exposure (R: P=0.34, L: P=0.14, ANOVA).

LT exposure decreased the skin temperatures of bothhind paws in all four groups of rats (mid). In the controland SYX rats, LT exposure decreased the temperature ofboth injured and uninjured paws to a similar level, i.e.,smaller ∆T during LT exposure (control-mid and SYX-mid). In the CCI and SYX+CCI rats, on the contrary, LTexposure caused a greater decrease in temperature on thenerve-injured side, i.e., significant expansion in ∆T(CCI: P=0.002, SYX+CCI: P=0.014, paired t-test).Therefore, the mid-∆Ts of both CCI and SYX+CCI rats,but not SYX rats, were significantly different from thatof the controls (P<0.05 for both, ANOVA with a posthoc analysis). Ninety minutes after returning to the nor-mal ambient temperature (post), the skin temperature ofboth hind paws in the control and CCI rats had returnedto the pre-exposure level. In the sympathectomizedgroups, the skin temperature of both hind paws after re-turning to normal temperature (post) was higher than thepre-exposure levels (post vs pre, P<0.05 for all groups,ANOVA with a post hoc analysis).

Discussion

CCI in rats has been reported to produce symptoms in-cluding allodynia and hyperalgesia to mechanical orthermal stimulations, and spontaneous pain, all of whichapproximate the clinical features of human neuropathicpain (Bennett and Xie 1988; Attal et al. 1990). These ab-normal pain-related behaviors in CCI rats were con-firmed in the present study. LT exposure induced the fol-lowing changes in the CCI rats but not the control rats:

(1) a decrease in the withdrawal threshold to VFH andpaw pressure stimulations (mechanical allodynia), (2) anincrease in the withdrawal duration to pinprick stimula-tion (mechanical hyperalgesia), and (3) a prolongation ofthe cumulative duration of foot lifting (increase in spon-taneous pain). These results indicate that lowering theambient temperature by 7°C can aggravate behavioralabnormalities displayed in a model of painful neuropa-thy. These changes are similar to those effected by low-pressure exposure (Sato et al. 1999), and may accountfor the aggravation of pain in neuropathic patients duringexposure to a mildly cold environment (Ochoa andYarnitsky 1994).

In the present study, CCI surgery did not induce anychanges in the VFH thresholds of four out of ten nerve-injured rats on the day of LT exposure. Moreover, CCIsurgery failed to induce obvious changes in the with-drawal threshold to the paw pressure stimulation onPOD 26, as compared with the sham operation. Consid-ering these results, there may be some question as towhether our CCI procedure successfully produced me-chanical allodynia in all rats injured. In fact, there is fair-ly general agreement that not all CCI rats with hyperal-gesia show obvious mechanical allodynia. Nonetheless,we exposed all of the nerve-injured rats to the LT envi-ronments in the present study, and interestingly, such ex-posure reduced the mechanical thresholds of rats bothwith and without allodynia before exposure. The presentresults thus demonstrated that LT exposure not only ag-gravated the mechanical allodynia in the CCI rats, butalso unmasked it in some apparently normal CCI rats.

In marked contrast to low-pressure exposure studies(Sato et al. 1999), lumbar sympathectomy preceding theconstriction injury did not prevent the aggravations ofmechanical allodynia and hyperalgesia induced by LTexposure. These results, therefore, suggest a fundamentaldifference in the mechanisms by which low pressure andLT exposures aggravate pain-related behaviors. They

Table 1 Change in hind pawtemperature due to low-temper-ature (LT) exposure. Skin tem-peratures of right (R, injuredside) and left (L, uninjuredside) hind paws, and differencebetween both sides (∆T) before,during, and after LT exposureare shown (°C, mean ± SEM).(CCI Chronic constriction in-jured rats, SYX sympathectomi-zed rats, SYX+CCI sympathec-tomized CCI rats, pre beforeLT exposure, mid during LT ex-posure, post after LT exposure)

Pre Mid Post

Controla (n=9) R 28.0±0.5 20.5±0.8c 29.2±0.7L 28.0±0.5b 20.1±0.8c 29.1±0.7∆T 0.0±0.2 0.4±0.3 0.2±0.5

SYX (n=8) R 26.8±0.7 20.2±0.5c 29.6±0.4c

L 26.6±0.5b 20.1±0.8c 29.1±0.4c

∆T 0.2±0.3 0.1±0.4 0.5±0.2CCI (n=14) R 27.1±0.5 18.5±0.5c 27.6±0.6

L 26.7±0.5b 19.4±0.5c 27.5±0.6∆T 0.3±0.2 –0.9±0.3d 0.1±0.1

SYX+CCI (n=13) R 25.8±1.2 19.9±1.1c 29.7±0.3c

L 25.4±1.1b 20.6±1.1c 29.8±0.4c

∆T 0.4±0.3 –0.6±0.3d –0.1±0.3

a Control: sham-operated ratsb Differences between R and L before LT exposure (pre) are not significant (paired t-test)c Significantly different from each pre-exposure value (P<0.05 for all, repeated measures ANOVAfollowed by a post hoc analysis)d LT exposure caused a greater decrease in R temperature (mid), i.e., significantly different from thatof the control (P<0.05 for both, ANOVA with a post hoc analysis)

would seem at first to indicate that sympathetic nerve ac-tivity does not play a significant role in the aggravatingeffect of LT exposure. This, however, is highly unlikely,since exposure to a cold environment is known to be as-sociated with activation of the sympathetic nervoussystem (indicated by increasing skin or muscle sympa-thetic nerve activities and other effects; Delius et al.1972a,b; Bini et al. 1980; Okamoto et al. 1994) and anincrease in plasma norepinephrine levels (Benedict et al.1977). These facts lead us to speculate that there is someother mechanism involved which masks the effects ofsympathetic nerve activities. We can propose three hy-potheses to account for such a mechanism.

First, decreased local skin temperature during LT ex-posure may increase pain. It is likely that such a processwould act through a mechanism that works only innerve-injured rats, since augmentation of pain-relatedbehaviors during LT exposure was found only in thenerve-injured rats, even though skin temperature was de-creased to a similar level in all four groups of rats. In thenerve-injured rats, lowered skin temperature might haveactivated the peripheral nociceptive and/or mechanore-ceptive fibers in the injured sciatic nerve and augmentedtheir responsiveness to mechanical stimuli, resulting inaugmented mechanical allodynia and hyperalgesia. Sucha direct action on sensory receptors could be powerfulenough to mask the augmenting effect of sympathetic ac-tivities. Supporting this hypothesis are the observationsof: (1) spontaneous discharge rates in injured afferentC-fiber endings in experimental nerve-end neuroma(Matzner and Devor 1987) and (2) increased activity ofmechanoreceptors by cooling (Burton et al. 1972;Duclaux and Kenshalo 1972; Booth and Haiin 1974).Whether or not LT exposure alters afferent nerve activi-ties in the injured sciatic nerve remains to be clarified infuture experiments. A similar direct effect of cooling onmicrovessels could be hypothesized, since LT exposuredecreased skin temperature of the injured hind paw to alarger degree than the control side (significant expansionin ∆T) in both CCI and SYX+CCI rats.

A second possible explanation is that humoral cate-cholamines, released from the adrenal medulla as a resultof the cold environment, might contribute to a greaterextent than do sympathetic catecholamines. It is likelythat sympathetic outflow to the adrenal medulla survivedthe sympathectomy in the present study (Araki et al.1984). Assuming this hypothesis to be true, the cate-cholamines released from the adrenal medulla couldhave induced either of the following two processes.First, the humoral catecholamines could provoke vaso-constriction, resulting in decreases in the local tempera-ture and ischemia which induce the aggravation of pain-related behaviors in the nerve-injured rats through a pro-cess similar to that described in the first hypothesis. Asecond possibility is that humoral catecholamines acti-vated and sensitized nociceptive fibers in the injured sci-atic nerve (O'Halloran et al. 1996). Previous studies havedemonstrated the existence of a vasoconstrictor mecha-nism, which is based on the sympathetic denervation su-

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persensitivity of microvessels to circulating catechola-mines in CCI rats (Wakisaka et al. 1994; Kurvers et al.1996). In the present experiment, LT exposure caused agreater decrease in skin temperature in the injured hindpaw than on the control side in the nerve-injured rats, al-though the difference was much smaller than the abso-lute decrease in skin temperature. It is, therefore, likelythat these differences, brought about by released cate-cholamines, depend on sympathetic denervation super-sensitivity. However, LT exposure did not influence theR-L temperature difference (∆T) in the SYX rats, where-as it increased it in the SYX+CCI rats. These results,therefore, suggest that the supersensitivities of microves-sels or nociceptive fibers to catecholamines are not gen-erated by a disturbance of sympathetic outflow causedby CCI, but rather may be caused by other processes re-lated to CCI (occlusion of the endoneural vessels, in-flammation, etc.). Similar abnormal responses to cate-cholamines in C-fiber polymodal receptors were seen inadjuvant-induced inflamed or streptozotocin-induced di-abetic rats as well as nerve-injured animals (Sato andPerl 1991; Sato et al. 1993; O'Halloran et al. 1996; Satoand Kumazawa 1996). Considering these previous stud-ies, there is no doubt that these humoral mechanisms in-volve the upregulation of adrenoceptors in skin micro-vessels or nociceptive fibers (Nishiyama et al. 1993),and thus they would both become more excitable and re-spond directly to endogenous catecholamines.

A third hypothesis is that LT exposure activates non-neuronal cells, such as mast cells, which might then re-lease algesic substances (Juhlin and Shelly 1961;Ringkamp et al. 1994) that heighten nociceptor activities(Lang et al. 1990; Mizumura et al. 1994; Koda et al.1996; Mizumura and Kumazawa 1996). The exact mech-anism by which this would occur remains open to study.

In conclusion, the present experiment indicated thatexposure to simulated low ambient temperature aug-mented/induced mechanical allodynia and hyperalgesia,as well as spontaneous pain in a model of painful neu-ropathy. These augmenting effects were similar to thoseof low-pressure exposure. Based on the different effectsof sympathectomy with LT and low-pressure exposures,we also suggest there may be other factors in addition tosympathetic ones, for which we have proposed threepossible mechanisms. Such factors may play a signifi-cant role in these augmenting effects, and mask the ef-fects of sympathetic nerve activity.

Acknowledgements We wish to thank Dr. K. Koga and Mr. Y.Ohta for their technical assistance. This work was partly supportedby Grants-in-Aid for Scientific Research from the Japanese Minis-try of Education, Science and Culture, and the Kato Ryutaro Foun-dation at Nagoya University. Additional support was received bythe Ground Research for Space Utilization program promoted byNASDA and the Japan Space Forum.

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