Animal handling and the experimental protocol were approved by the Institutional Bioethical Committee (UC-FCS-DIPI-29102012AE/ JM) following both the US National Institute of Health guide for the care and use of laboratory animals (NIH) and local regulations [15].

Adult male Sprague-Dawley strain rats weighing 300-350 g (n = 12) were housed in a low stress environment (12:12 h light: dark schedule) with food and water ad libitum. After one week of acclimatization, animals were anesthetized with thiopental (60 mg$ \cdot $Kg$ ^{-1} $; i.p.). Anesthesia was verified by absence of paw flexor and corneal reflexes during the experiment. Atropine was given subcutaneously (0.25 mg diluted in 0.5 ml NaCl 0.9%) to reduce secretions into the respiratory tract. Colorectal temperature was constant at 34; 37 or 40 $\pm$ 0.5 $^{\circ}$ C (mean $ \pm $ SD) by a feedback controlled heating blanket underneath the ventral surface of the animal. Laminectomies (T$ {}_{10} $ - L$ {}_{3} $) were performed to expose the lumbar enlargement of the spinal cord that was then covered with tempered mineral oil (body temperature $\pm$ 0.5$^{\circ}$C). The left hind paw sural nerve was dissected free and covered with tempered mineral oil. The paws were left in close contact with the heating blanket to maintain body temperature. In some experiments intracarotid blood pressure monitoring was performed to confirm that while under anesthesia, neither the electrical nor the HNS induced blood pressure increases.

Rats were placed in a stereotaxic frame for CDPs to be ipsilaterally recorded by a metal electrode with an uninsulated tip (250 $ \mu $m $ \varnothing $, $ < $ 50 $ \Omega $ steel filaments) orthodromically activated by electrical stimulation of the left hind paw sural nerve (hook bipolar electrode, 5 mA; 0.5 ms; 0.05 Hz; A\&M System A\&M System^TM Mod 1700). The reference electrode was placed under the abdominal skin. The stimulation intensity was set to 10 times the threshold of N-wave induction [16] and activated both A and C fibers [17]. This was sufficient to elicit a light contraction of the hind paw. CDPs were amplified ($ \times $ 1000; A\&M System^TM), band-pass filtered (10 Hz-500 Hz), digitized at 14.4 kHz (DATAQ^TM), supervised on-screen (WinDaq/XL^TM), and saved to hard disk. Amplitude (from basal isoelectric line) and duration (time difference between isoelectric line crossing points) of N- and P-waves were measured off-line by means of digital cursors from a five-wave average using Data View Software [18].

HNS to elicit DNIC mechanisms consisted of applying a clamp that sequentially exerted a noxious pressure of 4.0 kg/cm² for 5 min [18] to different body areas, i.e. tail, right hind paw, right forepaw, muzzle, and left forepaw. The clamp was previously tested on an investigator’s finger.

A total of 12 rats were studied and their results analyzed. CDPs were recorded for five minutes during the control condition (CC), i.e. sural nerve stimulation only. Thereafter, maintaining sural nerve stimulation, HNS was applied for five minutes sequentially in the tail, right hind paw, right forepaw, muzzle, and left forepaw, with five minutes of CC recording after each HNS application. This duration has been reported sufficient for recovery of basal activity in SDH neurons [4,11]. The HNS body site sequence was applied randomly clockwise or anticlockwise to every subject for each temperature (34; 37 and 40$ ^{\circ} $C), which was also randomly increased or decreased prior to each application. Wave values were analysed only after two minutes of each condition. Recordings were started after the test temperature had been changed for 15 minutes, or about 5 to 6 minutes after subjects reached a stable core temperature.

Results were obtained from the average of five N- and P-waves. Wave amplitudes ($ \mu $V) and duration (ms) were expressed as mean $ \pm $ SD. Data were normalized as a percentage of control condition values. Two rounds of HNS application were analyzed for each subject and averaged for each body site. Thermo-dependence was evaluated by Q$ {}_{10} $ values for 34-37$ ^{\circ} $C and for 37-40$ ^{\circ} $C which were linearly extrapolated for 10$ ^{\circ} $C for N- and P-wave amplitude and duration. Differences between pairs (34 to 37$ ^{\circ} $C; 37 to 40$ ^{\circ} $C and 34 to 40$ ^{\circ} $C) or groups (34, 37 and 40$ ^{\circ} $C) were analyzed by Wilcoxon sign-rank or Kruskal-Wallis tests, respectively. Data dispersion induced by HNS was evaluated by coefficient of variation Fligner-Killeen test. A p < 0.05 was considered significant for all analyses. Analysis employed the PAST statistical package [19].

Acute pain and inflammatory soup enzymatic activity i.e. cyclooxygenases, phospholipases C and A, and lipoxygenase, among many others, display similar or even greater thermo-dependence [3, 24]. These enzymes increase their activity with temperature as well as the concentration of their products which are components of the inflammatory soup, inducing neuronal excitation. Noxious mechanical stimulation similar to that used in the present study was able to induce increased pain-related enzymatic activation which increased with temperature not only at the stimulus application body sites but also in the spinal dorsal horn [24]. However, this increased activity should be expressed as increased nociception resulting in a higher activity of the descending pain modulation system. In contrast, results support the notion of an attenuated efficiency of this modulation at temperatures over 37$ ^{\circ} $C, hence, providing evidence for the participation of additional mechanisms to those of temperature-dependent enzymatic activation(see discussion below).

Hyperthermia activates the immune system, and immune cells in turn sensitize primary afferent neurons, increasing pain sensation. In fact, the cytokine IL6-COX2-PGE2 axis that drives fever [25] also plays a major role in nociception [20]. It is probable that the acute increase in temperature seen in these experiments, activates fast innate immune responses via phagocytic or cytotoxic effectors [25], but as discussed in the previous section, a locally increased response should increase pain modulation, but rather, results strongly suggest additional mechanisms which in turn attenuate modulation at higher temperatures.

At low temperature, deactivation of the voltage-gated sodium channel is less extreme than the activation process. This results in longer channel opening times and increased membrane depolarization and excitability. At temperatures around 42$ ^{\circ} $C, activation and deactivation processes accelerate, thus reducing both channel opening time and neuronal excitability. These effects tend to reduce activation signals coming into the SDH [26,27,28], but the inhibitory effect is probably less intense for lower temperature increases [28].

The dorsolateral funiculus is the main descending pathway for modulation of SDH. It exerts inhibitory and/or excitatory pain modulation through a balance between serotonergic (5-HT) and noradrenergic ($ \alpha _{2} $) receptor effects, respectively. This results in an algebraic effect [20,29,30], but descending modulation could not evoke a generally antinociceptive tone, instead excitatory and inhibitory systems may simultaneously be active and modulate SDH neuronal responses [31]. During resting conditions, HNS application moves this balance toward noradrenergic-mediated inhibition and away from serotonin-mediated excitation [21,32]. These two descending brain stem systems are interconnected and influence each other [21], resulting in a final balance at the SDH with tonic input from the rostral ventral medulla (RVM) system and phasic input from the subnucleous reticularis dorsalis (SRD) system [20,29]. Results reported here might indicate the expression of a differential thermo-dependence in the descending modulation to the SDH in which, maintaining the same stimulation conditions, increased temperature unbalances the descending effect toward serotonergic, over adrenergic, pathways and resulting in decreased HNS inhibition.

DNIC exerts also a differential control over A$ \beta $-fibers, and A$ \delta $- and C-fibers [33]. In this study, despite the fact that A$ \beta $-fibers were probably more activated at HNS sites due to the use of a noxious clamp than at the test site where the sural nerve was electrically stimulated, the N-wave was reduced at 40$ ^{\circ} $C, suggesting a weakening of DNIC inhibition. Additionally, small diameter dorsal root ganglia neurons show heat-induced depolarization at two different temperatures, above 42$ ^{\circ} $C with a Q$ {}_{10}$=$ 18$ to 27 due to activation of TRPV1 channels, and above 52$ ^{\circ} $C, due to TRPV2 channels. It should be noted that temperatures evaluated in the present study (34, 37 or 40$ ^{\circ} $C) certainly correspond to the animal body core, are far below the noxious threshold, and below the lowest activation threshold for both TRPV1 and TRPV2 channels.

The evaluated SDH response involves three main components: a) Primary afferents and SDH where the main noxious stimulus was applied and processed, e.g. sural nerve and segmental lumbar SDH, b) Distant body site with its primary afferents and corresponding segmental SDH, where HNS was applied and processed, and c) Supraspinal modulation centers and their descending pathways separated into two different functional subsystems. The first exhibits a phasic response at the SRD (DNIC), the second shows a tonic response exerting noradrenergic- and opioid-mediated inhibition and serotonin-mediated excitation of SDH neurons [21,32]. In these results, reduced inhibitory effects by temperature increases may be the result of two possible conditions, the first, increased activity at the noxious stimulation test site, i.e. sural nerve pathway or at the lumbar SDH neuronal population to a constant intensity stimulus. This option seems less probable as the same increased activity should occur at the HNS application sites with the same temperature changes i.e. fore and hind paws. The second situation concerns HNS application sites, especially the tail, where lesser skin temperature changes with the lowest changes in inhibitory effect were observed and enhanced the role of temperature in peripheral tissue for nocifensive responses.

SDH excitability of A$ \delta $ and C-fibers activated by an intense sural nerve electrical stimulation could be enhanced at 40$ ^{\circ} $C due to their expression of TRPV3 and TRPV4 channels. This results in an increased incoming signal from the test site (sural) overcoming a smaller increase in HNS-induced descending inhibition, which at its site was mostly activated through A$ \beta $ fibers with a low expression of TRP channels [12,13]. During increased temperature, the HNS signal to brainstem modulation centers remains relatively the same or shows a small increase, but the signal at the test noxious site shows relatively increased activity, therefore, the balance of descending modulation shifts towards reduction of the inhibitory effect. A shift in the balance of descending modulation towards a reduction of adrenergicopiod-mediated inhibition and/or increased 5HT excitation is also possible. Excluding HNS applied to the tail (vide supra), this misbalance could not be generated by a reduced activity of the sensory process from the HNS site because it shares the same temperature as the test site. As a result of this misbalance at the SDH, increased neuronal excitability, number of activated interneurons, and discharge synchronization to incoming sensory input, are expressed by increased amplitude and duration of the N-wave [7,10,11,16].

In contrast with the N-wave, the P-wave did not show thermal dependence through amplitude and duration changes. It is ruled by various mechanisms and processes more complicated than those governing the N-wave [7, 11, 16, 20]. The P-wave could show changes such as increased coefficient of variation of amplitude or duration [11], but the temperatures tested did not induce such P-wave responses. Complexity of the P-wave origin and its different response as compared with the N-wave requires further investigation. However, one of the reported results could be related to this point when in absence of HNS, changes in body temperature did not modify either N- or P-wave amplitude and duration. This suggests that thermo-dependent effects are mainly exerted through the phasic part of the descending pain modulation system i.e. from the subnucleus reticularis dorsalis system [29,34].

Results of the present study strongly suggest that increased body temperature within the clinical range of about 3$ ^{\circ} $C i.e. fever, could increase pain sensation by reduction of HNS-induced inhibition. This conclusion contributes to understanding of the behavior of the clinically ill and may explain the low pain threshold and hyperalgesia shown by subjects during fever [35, 36]. Additionally, experimental evidence is provided for weakened pain regulation during increased body temperature, which is present in the majority of inflammation cases [36]. Hypothermia could induce the opposite response for fever, decreasing pain sensation by enhancement of HNS-induced inhibition at SDH additional to its enzymatic slow-down effect.